Monoclonal antibody capable of binding to anexelekto, and use thereof

ABSTRACT

The present inventors have succeeded in producing anti-AXL antibodies with specific functions. The present inventors also discovered that the antibodies have an angiogenesis-suppressive effect and an antitumor effect, and thereby completed the present invention. The anti-AXL antibodies of the present invention are useful as angiogenesis inhibitors and agents for inducing or inhibiting phosphorylation activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/742,947, having a 371(c) date of Oct. 1, 2010, which is theNational Stage of International Application Serial No.PCT/JP2008/070739, filed on Nov. 14, 2008, which claims the benefit ofJapanese Application Serial No. 2007-297168, filed on Nov. 15, 2007. Thecontents of the foregoing applications are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to monoclonal antibodies that bind toanexelekto, agents containing the antibodies as an active ingredient,and methods for using the antibodies.

BACKGROUND

Anexelekto (also referred to as “AXL”, “UFO”, “ARK”, or “TYRO7”;hereinafter referred to as “AXL”), which has been cloned from patientswith chronic myeloid leukemia, is an oncogene capable of transformingmouse NIH3T3 cells when highly expressed (Non-patent Documents 1 and 2).AXL protein is a 140-kDa receptor tyrosine kinase (Non-patent Document3), and is said to be responsible for signal transduction to downstreammolecules through its autophosphorylation, which occurs after it bindsto the ligand Gas6 (growth arrest specific gene 6) (Non-patent Document4). Receptor tyrosine kinases, such as Sky, Mer, and AXL, are known asreceptor tyrosine kinases with Gas6 as a ligand (Non-patent Document 5).

AXL is presumed to have molecular functions involved in cell growthenhancement, suppression of apoptosis, cell migration, and celladhesion. Experimentally observed phenomena in cells treated with Gas6protein support this presumption. Reported experimental results includeits suppression of cell death and its enhancement of cell growth in ratvascular smooth muscle (Non-patent Documents 6 and 7), the accelerationof cell growth and the suppression of cell death after serum starvationin mouse NIH3T3 cells (Non-patent Documents 8 and 9), the promotion ofcell growth in mouse cardiac fibroblasts (Non-patent Document 10), theenhancement of cell growth in human prostate cancer cells (Non-patentDocument 11), the enhancement of cell growth and infiltration and thesuppression of cell death in human gastric carcinoma cells (Non-patentDocument 12), the enhancement of the migration ability of human and ratvascular smooth muscle cells (Non-patent Document 13), the enhancementof the cell migration ability of mouse neurons (Non-patent Document 14),and the aggregation of cells highly expressing mouse AXL (Non-patentDocument 15).

Similarly, PI3K-Akt pathway and MAPK pathway are said to function asdownstream pathways of the signal transduction mediated by AXL based onmolecular analyses of intracellular signals after treatment with Gas6(Non-patent Document 5). An analysis with a yeast two-hybrid methodusing an AXL intracellular region as the bait confirmed the directmolecular interactions with these downstream pathways. As a result,GrbB2/PI3K/p55γ/SOCS-1/NcK2/RanBP2/C1-TEN were identified (Non-patentDocument 16). The interactions of these molecules suggest the presenceof intracellular signal transduction pathways as downstream from the AXLsignal. Furthermore, the observed interactions support the presumptionthat AXL functions in cell growth enhancement, the suppression ofapoptosis, cell migration, and cell adhesion. AXL has also beenidentified as a gene highly expressed when TNFα-induced cell death ofmouse fibroblasts is suppressed by IL-15. The suppression ofTNFα-induced cell death was abolished by suppressing AXL expression, andthe phosphorylation of IL-15 receptors and AXL was enhanced by treatmentwith IL-15. These experimental findings also suggest that signaltransduction is mediated by the complex of AXL and IL-15 receptor(Non-patent Document 17).

Tumorigenicity of nude mice has been reported to dissipate as a resultof inhibiting Gas6-dependent phosphorylation of AXL in human gliomalines overexpressing the AXL dominant negative form (Non-patent Document18). However, there have been no reports or suggestions and remainunclear whether any anti-AXL antibody which inhibits phosphorylationexists.

AXL is a single-pass transmembrane receptor tyrosine kinase, and theextracellular region is composed of two immunoglobulin-like domains(referred to as IgD1 and IgD2) and two fibronectin type III domains(referred to as FND1 and FND2) from the N-terminal side (Non-patentDocument 3). Although FND is known to be expressed in molecules such asneural cell adhesion molecules and nephrins involved in intercellularadhesion, detailed functions of FND in AXL are unclear (Non-patentDocument 19).

AXL has been identified as an oncogene that retains an inherent abilityto transform cells, and has been studied as a carcinogenesis-relatedmolecule. Many analyses of AXL expression have been reported on theprotein and mRNA. The high expression of AXL protein has been reportedin human tumor tissues and tumor cells, including lung cancer(Non-patent Document 20), breast cancer (Non-patent Document 21),ovarian cancer (Non-patent Document 22), thyroid cancer (Non-patentDocument 23), melanoma (Non-patent Document 23), renal cancer(Non-patent Document 24), gastric cancer (Non-patent Document 12), andglioma (Non-patent Document 25). Furthermore, the high expression of AXLprotein is suggested by high levels of AXL mRNA in esophageal cancer(Non-patent Document 26), colon cancer (Non-patent Document 27), andacute myeloid leukemia (Non-patent Document 28). There are also reportsof the inhibition of lumen formation via the suppression of AXL by RNAiin HUVEC (Non-patent Document 29), the reduced tumor-forming ability ofhuman breast cancer cells in mice resulting from the constitutivesuppression of AXL (Non-patent Document 29), and the reducedtumor-forming ability of human glioma cells in mice resulting from theconstitutive high expression of dominant negative mutants (Non-patentDocument 25). The involvement of AXL molecular functions in tumor growthis strongly suggested.

Polyclonal antibodies to the extracellular domain of AXL have beenreported to have a migration inhibitory action on highly invasive breastcancer cell lines (Patent Document 1). However, non-clinical studiesshowed that drugs demonstrating cancer-cell-migration-inhibitory actiondo not necessarily demonstrate antitumor activity. For example, matrixmetalloproteinase (hereinafter abbreviated to “MMP”) has been known topromote tumor infiltration and migration. Thus, attention has beenfocused on various matrix metalloproteinase inhibitors that inhibit theenzyme activity of MMP, and clinical studies have been conducted onvarious pharmaceutical agents such as Batimastat, Marimastat, andPrinomastat. However, antitumor effects have not been observed in theclinical trials (Non-patent Document 30).

Accordingly, there have been no reports or suggestions and it remainsunknown whether anti-AXL antibodies have antitumor effects particularlyin vivo, whether they can suppress angiogenesis, and whether they cansuppress cancer.

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DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

The objectives of the present invention are to provide anti-AXLantibodies and uses thereof. More specifically, the objectives of thepresent invention are to provide methods for inhibiting angiogenesisusing anti-AXL antibodies, methods for suppressing cell growth, methodsfor inhibiting AXL function, methods for accelerating AXL function, andmethods for reducing the AXL expression level. A further objective ofthe present invention is to provide anti-AXL antibodies with noveleffects.

Means for Solving the Problems

As a result of conducting dedicated studies, the present inventorssucceeded in producing anti-AXL antibodies with specific functions anddiscovered that these antibodies have an angiogenesis-suppressive effectand an antitumor effect, and they therefore completed the presentinvention. More specifically, the present invention includes:

[1] a monoclonal antibody that binds to AXL;[2] the antibody according to [1], which has cell-growth-suppressiveactivity;[3] the antibody according to [1], which suppresses cancer cell growth;[4] the antibody according to any of [1] to [3], which binds to FND1;[5] an antibody prepared using as an antigen a peptide comprising entireFND1 or a sequence comprising at least five or more consecutive aminoacids thereof;[6] the antibody according to any of [1] to [5], which has agonisticactivity for AXL;[7] the antibody according to any of [1] to [5], which has antagonisticactivity for AXL;[8] the antibody according to [7], which is obtained by selecting anantibody in which phosphorylated tyrosine is not detected in AXL whencontacting it to an AXL-expressing cell together with an AXL ligand;[9] the antibody according to any of [1] to [8], which has an activitythat reduces AXL expression level;[10] the antibody according to any of [1] to [9], which has angiogenesisinhibitory activity;[11] an antibody according to any of the following (a) to (j):(a) an antibody (Ax285) produced from a hybridoma deposited underAccession No. FERM BP-10858;(b) an antibody (Ax292) produced from a hybridoma deposited underAccession No. FERM BP-10859;(c) an antibody (Ax223) produced from a hybridoma deposited underAccession No. FERM BP-10853;(d) an antibody (Ax96) produced from a hybridoma deposited underAccession No. FERM BP-10852;(e) an antibody (Ax258) produced from a hybridoma deposited underAccession No. FERM BP-10856;(f) an antibody (Ax284) produced from a hybridoma deposited underAccession No. FERM BP-10857;(g) an antibody (Ax7) produced from a hybridoma deposited underAccession No. FERM BP-10850;(h) an antibody (Ax51) produced from a hybridoma deposited underAccession No. FERM BP-10851;(i) an antibody (Ax225) produced from a hybridoma deposited underAccession No. FERM BP-10854; and(j) an antibody (Ax232) produced from a hybridoma deposited underAccession No. FERM BP-10855;[12] an antibody that binds to the same epitope as an epitope bound byany of the antibodies according to [11];[13] an antibody that comprises a CDR sequence identical to a CDRsequence comprised in any of the antibodies according to [11];[14] an antibody in which sequences of heavy chain CDR1, 2, and 3 areSEQ ID NOs: 4, 5, and 6, respectively;[15] an antibody that comprises a heavy chain CDR comprising an aminoacid sequence of the heavy chain CDR of the antibody according to [14]with a substitution, deletion, insertion, and/or addition of one or moreamino acids, and is functionally equivalent with the antibody accordingto [14];[16] an antibody in which sequences of light chain CDR1, 2, and 3 areSEQ ID NOs: 8, 9, and 10, respectively;[17] an antibody that comprises a light chain CDR comprising an aminoacid sequence of the light chain CDR of the antibody according to [16]with a substitution, deletion, insertion, and/or addition of one or moreamino acids, and is functionally equivalent with the antibody accordingto [16];[18] the antibody according to any of [13] to [17] that is a chimericantibody;[19] the antibody according to any of [13] to [17] that is a humanizedantibody;[20] a hybridoma according to any of the following (a) to (j):(a) a hybridoma deposited under Accession No. FERM BP-10858 (Ax285);(b) a hybridoma deposited under Accession No. FERM BP-10859 (Ax292);(c) a hybridoma deposited under Accession No. FERM BP-10853 (Ax223);(d) a hybridoma deposited under Accession No. FERM BP-10852 (Ax96);(e) a hybridoma deposited under Accession No. FERM BP-10856 (Ax258);(f) a hybridoma deposited under Accession No. FERM BP-10857 (Ax284);(g) a hybridoma deposited under Accession No. FERM BP-10850 (Ax7);(h) a hybridoma deposited under Accession No. FERM BP-10851 (Ax51);(i) a hybridoma deposited under Accession No. FERM BP-10854 (Ax225); and(j) a hybridoma deposited under Accession No. FERM BP-10855 (Ax232);[21] an angiogenesis inhibitor that comprises an anti-AXL antibody as anactive ingredient;[22] the angiogenesis inhibitor according to [21], wherein the antibodyis an antibody according to any of [1] to [19];[23] a cell-growth suppressant that comprises an anti-AXL antibody as anactive ingredient;[24] the suppressant according to [23], wherein the cells are cancercells;[25] the suppressant according to [23], wherein the antibody is anantibody according to any of [1] to [19];[26] the suppressant according to [23], wherein the anti-AXL antibody isan antibody that binds to FND1;[27] the suppressant according to [23], which comprises as an activeingredient an antibody that binds to IgD2 and has aphosphorylation-inhibition activity;[28] an AXL phosphorylation activity inducer, which comprises ananti-AXL antibody as an active ingredient;[29] the inducer according to [28], wherein the anti-AXL antibody is anantibody that binds to IgD;[30] the inducer according to [28], wherein the antibody is an antibodyaccording to [6];[31] an AXL phosphorylation activity inhibitor, which comprises ananti-AXL antibody as an active ingredient;[32] the inhibitor according to [31], wherein the anti-AXL antibody isan antibody that binds to IgD2;[33] the inhibitor according to [31], wherein the antibody is anantibody according to [7] or [8];[34] an agent that reduces an AXL expression level, which comprises ananti-AXL antibody as an active ingredient;[35] the agent for reducing an expression level according to [34],wherein the anti-AXL antibody is an antibody that binds to FND1;[36] the agent that reduces the expression level according to [34],wherein the antibody is an antibody according to [9];[37] a method for inducing phosphorylation of AXL using an anti-AXLantibody;[38] a method for reducing an AXL expression level using an anti-AXLantibody;[39] a method for inhibiting the phosphorylation of AXL using ananti-AXL antibody;[40] an anti-cancer agent that comprises an anti-AXL antibody as anactive ingredient;[41] the anti-cancer agent according to [40], wherein the antibody is anantibody according to any of [1] to [19];[42] The anti-cancer agent according to [40], which comprises as anactive ingredient an antibody that binds to IgD2 and has aphosphorylation-inhibition activity;[43] the anti-cancer agent according to [40], wherein the cancer ispancreatic cancer, gastric cancer, lung cancer, osteosarcoma, coloncancer, prostate cancer, melanoma, endometrial cancer, ovarian cancer,uterine leiomyoma, thyroid cancer, cancer stem cell, breast cancer,bladder cancer, renal cancer, glioma, neuroblastoma, or esophagealcancer;[44] the anti-cancer agent according to [42], wherein the cancer isglioma, gastric cancer, endometrial cancer, non-small-cell lung cancer,pancreatic adenocarcinoma, or breast cancer;[45] the anti-cancer agent according to [43], wherein the cancer ispancreatic adenocarcinoma or breast cancer;[46] the antibody according to [1], which has an AXLphosphorylation-inhibition activity;[47] a method for inhibiting angiogenesis using an anti-AXL antibody;[48] a method for using an anti-AXL antibody in manufacturing anangiogenesis inhibitor;[49] a method for suppressing cell growth using an anti-AXL antibody;[50] a method for treating and/or preventing cancer using an anti-AXLantibody;[51] a method for using an anti-AXL antibody in manufacturing acell-growth suppressant;[52] a method for using an anti-AXL antibody in manufacturing ananti-cancer agent;[53] a method for using an anti-AXL antibody in manufacturing aphosphorylation inducer;[54] a method for using an anti-AXL antibody in manufacturing aphosphorylation inhibitor;[55] a method for using an anti-AXL antibody in manufacturing an agentfor lowering the AXL expression level; and[56] a method for producing an anti-AXL specific antibody comprising:(a) immunizing a non-human animal with a peptide comprising entire FND1or a sequence comprising at least five or more consecutive amino acidsthereof; and(b) collecting an antibody from the non-human animal of (a) orcollecting an antibody-producing cell to collect an antibody produced bythe antibody-producing cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax292) of the present invention, in inducing AXLphosphorylation in cancer cells. The antibody was shown to induce thephosphorylation of a kinase domain of AXL.

FIG. 1B is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax258) of the present invention, in inducing AXLphosphorylation in cancer cells. The antibody was shown to induce thephosphorylation of a kinase domain of AXL.

FIG. 1C is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax285) of the present invention, in inducing AXLphosphorylation in cancer cells. The antibody was shown to induce thephosphorylation of a kinase domain of AXL.

FIG. 1D is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax223) of the present invention, in inducing AXLphosphorylation in cancer cells. The antibody was shown to induce thephosphorylation of a kinase domain of AXL.

FIG. 1E is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax96) of the present invention, in inducing AXLphosphorylation in cancer cells. The antibody was shown to induce thephosphorylation of a kinase domain of AXL.

FIG. 2A is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax51) of the present invention, in inhibiting ligand-dependentphosphorylation of AXL in a cell. The antibody was shown to inhibit theligand-dependent phosphorylation of a kinase domain of AXL.

FIG. 2B is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax7) of the present invention, in inhibiting ligand-dependentphosphorylation of AXL in a cell. The antibody was shown to inhibit theligand-dependent phosphorylation of a kinase domain of AXL.

FIG. 3A is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax292) of the present invention, in inducing AXLdownmodulation in cancer cells. The antibody was shown to induce thedownmodulation of AXL protein.

FIG. 3B is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax284) of the present invention, in inducing AXLdownmodulation in cancer cells. The antibody was shown to induce thedownmodulation of AXL protein.

FIG. 3C is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax285) of the present invention, in inducing AXLdownmodulation in cancer cells. The antibody was shown to induce thedownmodulation of AXL protein.

FIG. 3D is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax223) of the present invention, in inducing AXLdownmodulation in cancer cells. The antibody was shown to induce thedownmodulation of AXL protein.

FIG. 3E is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax7) of the present invention, in inducing AXL downmodulationin cancer cells. The antibody was shown to induce the downmodulation ofAXL protein.

FIG. 3F is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax225) of the present invention, in inducing AXLdownmodulation in cancer cells. The antibody was shown to induce thedownmodulation of AXL protein.

FIG. 3G is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax96) of the present invention, in inducing AXL downmodulationin cancer cells. The antibody was shown to induce the downmodulation ofAXL protein.

FIG. 3H is a photograph showing the activity of an anti-AXL monoclonalantibody (Ax258) of the present invention, in inducing AXLdownmodulation in cancer cells. The antibody was shown to induce thedownmodulation of AXL protein.

FIG. 4 is a drawing and photograph showing the activity of the anti-AXLmonoclonal antibodies of the present invention (Ax232, Ax292, Ax285, andAx284) in inhibiting in vitro angiogenesis. The antibodies were shown tohave an inhibitory activity of in vitro angiogenesis.

FIG. 5 is a drawing showing the antitumor effects of the anti-AXLmonoclonal antibodies of the present invention (Ax223, Ax285, Ax96,Ax292, Ax258, Ax7, Ax51, Ax284, and Ax225) in a mouse xenograft model ofhuman pancreatic adenocarcinoma.

FIG. 6 is a table showing the antitumor effects of anti-AXL monoclonalantibodies of the present invention (Ax223, Ax285, Ax96, Ax292, Ax258,Ax7, Ax51, Ax284, and Ax225) (2) in a mouse xenograft model of humanpancreatic adenocarcinoma, and summarizing the binding domain andphosphorylation-inhibiting activity of each antibody.

FIG. 7 is a drawing showing the antitumor effects of an anti-AXLmonoclonal antibody (Ax225) of the present invention in a mousexenograft model of human breast cancer.

DETAILED DESCRIPTION

A novel anti-AXL antibody is provided by the present invention.Moreover, a novel use of the anti-AXL antibody is provided by thepresent invention.

There are no particular limitations on the anti-AXL antibody of thepresent invention so long as it binds to AXL, and there are also noparticular limitations on its origin (such as human, mouse, rat, rabbit,or chicken), type (polyclonal antibody or monoclonal antibody), form(such as an altered antibody, modified antibody, antibody fragment, orminibody [low-molecular-weight antibody]), or such. Although there areno particular limitations on the anti-AXL antibody of the presentinvention, the antibody preferably specifically binds to anexelekto andis preferably a monoclonal antibody.

The anti-AXL antibody of the present invention also preferably has acell-growth-suppressive activity.

A preferable embodiment of the anti-AXL antibodies of the presentinvention is anti-AXL antibodies binding to FND1.

As is clear from the Examples described later, antibodies that bind toFND1 in particular have significantly high in vivo antitumor activitycompared to those of other antibodies.

Binding activity of anti-AXL antibodies to FND1 can be evaluated by amethod known to those skilled in the art, for example, the methodsdescribed below. Binding activity of anti-AXL antibody to FND1 isconfirmed by electrophoresing FND1 and western blotting with anti-AXLantibody.

An anti-AXL antibody with agonistic activity for AXL is an example ofthe preferable embodiment of the anti-AXL antibody of the presentinvention. An anti-AXL antibody with agonistic activity for AXL refersto the induction of phosphorylation mediated by AXL, and particularly tothe induction of the phosphorylation reaction of tyrosine, when theanti-AXL antibody binds to AXL. Although there are no particularlimitations on the target of the phosphorylation reaction that isinduced by the anti-AXL antibody with agonistic activity, an exampleincludes the autophosphorylation of AXL.

Whether or not an anti-AXL antibody has agonistic activity can bedetermined with a method known by those skilled in the art, for example,by the method described below. A test anti-AXL antibody is contactedwith cells expressing AXL (such as Calu-1, MDA-MB-231, or DU-145 cells),and AXL is subsequently extracted from the cells. The tyrosine in theextracted AXL is confirmed to be phosphorylated using ananti-phosphotyrosine antibody. More specifically, an anti-AXL antibodycan be confirmed as having an agonistic activity with the methodsdescribed in the Examples.

Examples of anti-AXL antibodies with agonistic activity include theantibodies (a) to (g) below:

(a) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10858 (Ax285);(b) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10852 (Ax96);(c) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10856 (Ax258);(d) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10859 (Ax292);(e) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10853 (Ax223);(f) an antibody recognizing the same epitope as the epitope recognizedby an antibody of any one of (a) to (e); and(g) an antibody with a CDR sequence identical to the CDR sequence of anantibody of any one of (a) to (e).

An antibody recognizing the same epitope as an antibody described abovecan be obtained according to, for example, the method described below.

Whether a test antibody shares an epitope with a certain antibody can beconfirmed by the competition of the two antibodies for the same epitope.Competition between antibodies is detected with a cross-blocking assayor the like. A competitive ELISA, for example, is a preferablecross-blocking assay. Specifically, in a cross-blocking assay, AXLprotein coated onto the wells of a microtiter plate is preincubated inthe presence or absence of the candidate competitive antibody, then ananti-AXL antibody, as indicated above, is added. The amount of theaforementioned anti-AXL antibody bound to the AXL protein in the wellsis indirectly correlated to the binding ability of the candidatecompetitive antibody (test antibody) competing for binding to the sameepitope. Thus, the greater the affinity of the test antibody for thesame epitope, the greater is the reduction in the amount of theaforementioned anti-AXL antibody bound to the wells coated with AXLprotein and the greater the increase in the amount of test antibodybound to the wells coated with AXL protein.

The amount of antibody bound to the wells can be measured easily bylabeling the antibody in advance. For example, a biotin-labeled antibodycan be measured using an avidin-peroxidase conjugate and a suitablesubstrate. A cross-blocking assay that uses an enzyme label such asperoxidase is specifically referred to as a competitive ELISA. Theantibody can be labeled with other labeling substances that can bedetected or measured. Specifically, radioactive labels and fluorescentlabels are known.

When the test antibody has a constant region derived from a speciesdiffering from that of the anti-AXL antibody indicated above, the amountof antibody bound to the wells can also be measured with a labeledantibody that recognizes the constant region of that antibody.Alternatively, when an antibody is derived from the same species but isof a different class, the amount of antibody bound to the wells can bemeasured with antibodies that recognize each class.

If a candidate competitive antibody can block the binding of theanti-AXL antibody by at least 20%, preferably by at least 20%-50%, andmore preferably by at least 50% compared with the binding activityachieved in a control test performed in the absence of the candidatecompetitive antibody, then the candidate competitive antibody is anantibody that substantially binds to the same epitope or that competesfor binding to the same epitope as the aforementioned anti-AXL antibody.

The determination of a CDR sequence to obtain an antibody with a CDRsequence identical to that of a certain antibody can be performed by oneskilled in the art according to known methods. For example, a CDRsequence can be determined by determining the full-length amino acidsequence of an antibody or the amino acid sequence of a variable region,and investigating its homology by applying the determined amino acidsequence to the database of antibody amino acid sequences developed byKabat et al. (“Sequence of Proteins of Immunological Interest”, US Dept.of Health and Human Services, 1983). The numbers in the framework andthe numbers in the CDR sequence can be determined according to thedefinition of Kabat (Kabat, A. E. et al., US Dept. of Health and HumanServices, US Government Printing Offices, 1991).

The full-length amino acid sequence of an antibody or the amino acidsequence of a variable region can be determined by one skilled in theart in accordance with known methods.

An antibody with a CDR sequence identical to that of a certain antibodymay have an identical sequence in at least one CDR of the six CDRs thatare present in the antibody. However, the antibody preferably has anidentical sequence in all three CDRs present in the heavy chain or anidentical sequence in all three CDRs present in the light chain, andeven more preferably, the antibody has an identical sequence in all sixCDRs present in the antibody.

Antibodies with a CDR sequence that is identical to a CDR sequence of acertain antibody include chimeric antibodies and humanized antibodies.Chimeric antibodies and humanized antibodies will be described below.

An example of another preferable embodiment of the anti-AXL antibody ofthe present invention is an anti-AXL antibody with antagonistic activityagainst AXL. An anti-AXL antibody with antagonistic activity against AXLrefers to an antibody with activity that inhibits the phosphorylationreaction mediated by AXL induced by the binding of an AXL ligand (suchas Gash) to AXL, and particularly the tyrosine phosphorylation reaction.The inhibition of the phosphorylation reaction can be carried out byinhibiting the binding between the AXL ligand and AXL, or by anothermethod. Although there are no particular limitations on the subjects ofphosphorylation inhibition reaction induced by an anti-AXL antibody withantagonistic activity, examples include the autophosphorylation of AXL.

Whether an anti-AXL antibody has antagonistic activity can be determinedby a method known to those skilled in the art, and for example, by themethod described below. A test antibody is contacted with cellsexpressing AXL (such as Calu-1, MDA-MB-231, or DU-145 cells) togetherwith an AXL ligand, and AXL is subsequently extracted from the cells.Phosphorylated tyrosine is confirmed not to be detected in the extractedAXL using an anti-phosphotyrosine antibody. More specifically, ananti-AXL antibody can be confirmed as having antagonistic activity usingthe methods described in the Examples.

Examples of anti-AXL antibodies with antagonistic activity includeantibodies (a) to (d) below:

(a) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10850 (Ax7);(b) an antibody produced from a hybridoma deposited under Accession No.FERM BP-10851 (Ax51);(c) an antibody recognizing the same epitope as the epitope recognizedby an antibody of any one of (a) to (b); and(d) an antibody having a CDR sequence identical to the CDR sequence ofan antibody of any one of (a) to (c).

An antibody recognizing the same epitope and an antibody with anidentical CDR sequence can be obtained with the methods previouslydescribed.

An antibody with antagonistic activity is useful for inhibitingangiogenesis, suppressing cell growth, and the like.

An example of another preferable embodiment of the antibody of thepresent invention is an antibody with activity that reduces the AXLexpression level. In the present invention, reducing the expressionlevel of AXL can indicate a reduction in the amount of AXL alreadypresent through the degradation of AXL or such, or can indicate areduction in the amount of newly expressed AXL by suppressing theexpression of AXL. Whether the AXL expression level has decreased can beconfirmed by a method known to those skilled in the art, and forexample, by the method described below. A test anti-AXL antibody iscontacted with cells expressing AXL (such as Calu-1, MDA-MB-231, orDU-145 cells), and the amount of AXL present in the cells issubsequently detected by immunoblotting or such. A comparison is thenmade between the amount of AXL detected when the test antibody iscontacted and the amount of AXL detected when the test antibody is notcontacted. More specifically, this can be confirmed according to methodsdescribed in the Examples.

Examples of anti-AXL antibodies with activity that reduces AXLexpression levels include antibodies (a) to (j) below:

(a) an antibody (Ax285) produced from a hybridoma deposited underAccession No. FERM BP-10858;(b) an antibody (Ax96) produced from a hybridoma deposited underAccession No. FERM BP-10852;(c) an antibody (Ax258) produced from a hybridoma deposited underAccession No. FERM BP-10856;(d) an antibody (Ax7) produced from a hybridoma deposited underAccession No. FERM BP-10850;(e) an antibody (Ax292) produced from a hybridoma deposited underAccession No. FERM BP-10859;(f) an antibody (Ax223) produced from a hybridoma deposited underAccession No. FERM BP-10853;(g) an antibody (Ax225) produced from a hybridoma deposited underAccession No. FERM BP-10854;(h) an antibody (Ax284) produced from a hybridoma deposited underAccession No. FERM BP-10857;(i) an antibody recognizing the same epitope as the epitope recognizedby an antibody of any one of (a) to (h); and(j) an antibody having a CDR sequence identical to the CDR sequence ofan antibody of any one of (a) to (i).

An antibody recognizing the same epitope and an antibody having anidentical CDR sequence can be obtained with the methods previouslydescribed.

An antibody with an activity that reduces the AXL expression level isuseful for inhibiting angiogenesis, suppressing cell growth, and thelike.

An example of another preferable embodiment of the antibody of thepresent invention is an antibody with an angiogenesis-inhibiting effect.Although there are no particular limitations on theangiogenesis-inhibiting effect of the present invention, so long as thenew formation of blood vessels is inhibited, examples include aninhibitory effect on the migration activity of vascular endothelialcells, an apoptosis-inducing effect on vascular endothelial cells, andan inhibitory effect on the vascular morphogenesis of vascularendothelial cells. A preferred example of an antibody with anangiogenesis-inhibiting effect is an antibody with anangiogenesis-inhibiting effect on tumor tissues. There are no particularlimitations on the tumor tissues, and examples include pancreatic cancertissue (such as pancreatic adenocarcinoma tissue), gastric cancertissue, lung cancer tissue (tissues of small-cell lung cancer,non-small-cell lung cancer, and such), osteosarcoma tissue, colon cancertissue, prostate cancer tissue, melanoma tissue, endometrial cancertissue, ovarian cancer tissue, uterine leiomyoma tissue, thyroid cancertissue, cancer stem cell tissue, breast cancer tissue, bladder cancertissue, renal cancer tissue, glioma tissue, neuroblastoma tissue, andesophageal cancer tissue. More preferable tissues are glioma tissue,gastric cancer tissue, endometrial cancer tissue, non-small-cell lungcancer tissue, pancreatic adenocarcinoma tissue, and breast cancertissue, particularly pancreatic adenocarcinoma tissue and breast cancertissue.

Whether or not an antibody has an angiogenesis-inhibiting effect can beconfirmed by a method known to those skilled in the art, and forexample, this can be confirmed using a commercially availableangiogenesis kit. More specifically, this can be confirmed with themethods described in the Examples.

Specific examples of antibodies with angiogenesis-inhibiting effectsinclude the previously described antibodies.

An example of another preferable embodiment of the antibody of thepresent invention is an antibody with cell-growth-suppressive activity.

Although there are no particular limitations on the cells whose growthis suppressed by the anti-AXL antibody, they are preferably cellsrelated to a disease, and more preferably cancer cells. When the cellsare cancer cells, there are no particular limitations on the type ofcancer, and examples include pancreatic cancer (such as pancreaticadenocarcinoma), gastric cancer, lung cancer (small-cell lung cancer,non-small-cell lung cancer, and such), osteosarcoma, colon cancer,prostate cancer, melanoma, endometrial cancer, ovarian cancer, uterineleiomyoma, thyroid cancer, cancer stem cell, breast cancer, bladdercancer, renal cancer, glioma, neuroblastoma, and esophageal cancer. Morepreferable cancers are glioma, gastric cancer, endometrial cancer,non-small-cell lung cancer, pancreatic adenocarcinoma, and breastcancer, particularly pancreatic adenocarcinoma and breast cancer.

There are no particular limitations on the mechanism for the suppressionof cell growth by the antibody of the present invention, and cell growthcan be suppressed by any mechanism, such as the inhibition ofangiogenesis, the inhibition of phosphorylation, the induction ofphosphorylation, or the reduction of the AXL expression level.

The following methods are preferably used to evaluate or measure thecell-growth-suppressive effects based on the anti-AXL antibody.

As a method of evaluating or measuring cell-growth-suppressive activityin vitro, a method is used in which the uptake by viable cells of[³H]-labeled thymidine added to their medium is measured as an indicatorof DNA replication ability. As a simpler method, a dye expulsion methodis used, in which the ability to exclude a dye such as Trypan Blueoutside from the cells is measured under a microscope, or an MTT methodis used. The latter uses the ability of living cells to convert MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), atetrazolium salt, to a blue formazan product. Specifically, a testantibody is added to the culture solution of the test cells togetherwith a ligand, and after a predetermined period of time, MTT solution isadded to the culture solution and this is left to stand for apredetermined period to allow the MTT to be incorporated into the cells.The MTT, which is a yellow compound, is converted to a blue compound bysuccinate dehydrogenase in the mitochondria of the cells. After thedissolution of this blue product and coloration, the absorbance ismeasured and used as an indicator of the number of viable cells. BesidesMTT, reagents such as MTS, XTT, WST-1, and WST-8 are commerciallyavailable (Nacalai Tesque, and such) and can be appropriately used. Acontrol antibody can also be used when measuring this activity.

A tumor-bearing mouse model can be used to evaluate or measure thecell-growth-suppressive activity in vivo. For example, after cancercells expressing AXL are transplanted into a non-human test animal,either intradermally or subcutaneously, a test antibody is administeredintravenously or intraperitoneally starting from the day oftransplantation or from the following day, either daily or at intervalsof a few days. The cell-growth-suppressive activity can be evaluated bymeasuring the tumor size over time. In a similar manner to theevaluation in vitro, cell-growth-suppressive activity can be determinedby administering a control antibody and observing whether the tumor sizein the anti-AXL antibody-administered group is significantly smallerthan the tumor size in the control antibody-administered group. When amouse is used as the non-human test animal, nude (nu/nu) mice can besuitably used, in which T-lymphocyte function has been lost due to agenetic deficiency in the thymus. The use of these mice makes itpossible to eliminate the involvement of T lymphocytes in the testanimal during the evaluation and measurement of thecell-growth-suppressive activity of the administered antibody.

Examples of anti-AXL antibodies with cell-growth-suppressive effectsinclude the previously described antibodies.

The anti-AXL monoclonal antibody of the present invention can beacquired using known methods. A monoclonal antibody derived from amammal is particularly preferable as the anti-AXL antibody of thepresent invention. Monoclonal antibodies derived from mammals includethose produced from hybridomas as well as those produced by a hosttransformed with an expression vector containing the antibody genes,using genetic engineering techniques.

A monoclonal-antibody-producing hybridoma can be generated using knowntechnology, such as that described below. First, AXL protein is used asthe sensitizing antigen for immunization according to ordinaryimmunization methods. Immune cells obtained from an immunized animal arethen fused with known parent cells, according to ordinary cell fusionmethods, to obtain hybridomas. A hybridoma that produces the anti-AXLantibody can be selected from these hybridomas by screening for cellsthat produce the target antibody using ordinary screening methods.

More specifically, monoclonal antibodies can be generated out accordingto, for example, the method described below. First, the AXL protein thatis used as the sensitizing antigen for obtaining the antibodies can beobtained by expressing the AXL gene. The nucleotide sequence of thehuman AXL gene is already known (GenBank Accession No. M76125). Afterinserting the gene sequence encoding AXL into a known expression vectorwith which to transform suitable host cells, the human AXL protein ofinterest can be purified from the host cells or from the culturesupernatant using known methods. Purified naturally occurring AXLprotein can also be used in the same manner. Purification can be carriedout by using several chromatographies, such as the usual ionchromatography and affinity chromatography, performed once or multipletimes, either in combination or alone. A fusion protein, in which thedesired partial polypeptide of the AXL protein is fused to a differentpolypeptide, can also be used as an immunogen. An antibody Fc fragmentor peptide tag, or the like, can be used to produce a fusion protein foruse as an immunogen. A vector that expresses a fusion protein can beproduced by fusing two or more types of desired genes encodingpolypeptide fragments in frame and inserting the fused genes into anexpression vector, as previously described. Methods of preparing fusionproteins are described in Molecular Cloning, 2nd edition (Sambrook, J.et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring HarborLaboratory Press, 1989). AXL protein purified in this manner can be usedas a sensitizing antigen for the immunization of a mammal.

A partial peptide of AXL can also be used as a sensitizing antigen.Examples of partial peptides of AXL include a peptide obtained bychemical synthesis from an amino acid sequence of human AXL, a peptideobtained by incorporating a portion of the human AXL gene into anexpression vector and expressing it, and a peptide obtained by degradinghuman AXL protein using a protease. There are no particular limitationson the region used as the partial peptide, and an extracellular regionof A×L, for example, can be used.

Moreover, a peptide having the sequence of entire FND1 or containing atleast its five consecutive amino acids can be preferably used as apartial peptide. Sequences containing at least five consecutive aminoacids refer to those preferably containing six or more and morepreferably eight or more consecutive amino acids. In addition, sequencescontaining at least five or more consecutive amino acids refer to aminoacid sequences having antigenicity.

There are no particular limitations on the mammal immunized with thesensitizing antigen. To obtain a monoclonal antibody by cell fusion, itis preferable to select an animal to be immunized after consideration ofits compatibility with the parent cells used for the cell fusion. Ingeneral, rodents are preferred as the immunized animal. Morespecifically, mice, rats, hamsters, or rabbits can be used for as theimmunized animal. Monkeys and the like can also be used as the immunizedanimal.

The animal described above can be immunized with a sensitizing antigenusing known methods. For example, in a typical method, the mammal isimmunized by injecting the sensitizing antigen intraperitoneally orsubcutaneously. Specifically, the sensitizing antigen is administered toa mammal several times every four to 21 days. The sensitizing antigen isused for immunization after dilution to a suitable dilution ratio withphosphate-buffered saline (PBS), physiological saline, or the like. Thesensitizing antigen can also be administered with an adjuvant. Forexample, it can be mixed with Freund's complete adjuvant and emulsifiedfor use as the sensitizing antigen. A suitable carrier can also be usedwhen immunizing with the sensitizing antigen. In particular, when apartial peptide with a low molecular weight is used as the sensitizingantigen, it is desirable to bind the sensitizing antigen to a carrierprotein, such as albumin, keyhole limpet hemocyanin, and the like, forimmunization.

After the mammal has been immunized in this manner and it has beenconfirmed that the level of the desired antibody in the serum hasincreased, the immune cells are harvested from the mammal and used forcell fusion. In particular, spleen cells can be used preferably as theimmune cells.

Mammalian myeloma cells are used as the cells to be fused with theimmune cells. The myeloma cells preferably have a suitable selectionmarker for screening. A selection marker refers to a trait that allowscells to live (or not) under certain culture conditions. Known selectionmarkers include hypoxanthine-guanine phosphoribosyl transferasedeficiency (hereinafter abbreviated to “HGPRT deficiency”) and thymidinekinase deficiency (hereinafter abbreviated to “TK deficiency”). Cellsdeficient in HGPRT or TK are hypoxanthine-aminoptering-thymidinesensitive (hereinafter abbreviated to “HAT sensitivity”). HAT-sensitivecells are unable to synthesize DNA and die in HAT selection medium.However, when fused with normal cells, they can continue to synthesizeDNA using the salvage pathway of normal cells and therefore they beginto grow in HAT selection medium.

HGPRT-deficient cells and TK-deficient cells can both be selected with amedium containing 6-thioguanine, 8-azaguanine (hereinafter abbreviatedto “8AG”) or 5′-bromodeoxyuridine. Although normal cells die as a resultof incorporating these pyrimidine analogs into their DNA, cellsdeficient in these enzymes are unable to incorporate these pyrimidineanalogs and can thus survive in the selection medium. A selection markerreferred to as G418 resistance also imparts resistance to2-deoxystreptamine-type antibiotics (gentamycin analogs) because it is aneomycin-resistance gene. Various myeloma cells that are suitable forcell fusion are known, and examples of myeloma cells that can be usedinclude P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550),P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81,1-7), NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6,511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8, 405-415),SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St.Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21), 5194(Trowbridge, I. S., J. Exp. Med. (1978) 148, 313-323), and 8210 cells(Galfre, G. et al., Nature (1979) 277, 131-133).

The fusion of the aforementioned immune cells and myeloma cells can becarried out according to known methods, such as the method of Kohler andMilstein (Kohler, G and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

More specifically, the aforementioned cell fusion can be carried out inan ordinary nutritive culture medium in the presence of a cell fusionpromoter. Examples of cell fusion promoters that can be used includepolyethylene glycol (PEG) and Sendai virus (HVJ). An auxiliary agent,such as dimethylsulfoxide, can also be added as desired to furtherenhance the fusion efficiency.

The ratio in which the immune cells and myeloma cells are used can beset arbitrarily. For example, there are preferably 1-10 times moreimmune cells than myeloma cells. Examples of culture media that can beused for the cell fusion described above include MEM and RPMI1640culture medium, preferably used for the growth of the aforementionedmyeloma cell lines, as well as ordinary culture medium used for thistype of cell culture. A serum supplement, such as fetal calf serum(FCS), can also be added to the culture medium.

Cell fusion is carried out to form target fused cells (hybridomas) bythoroughly mixing predetermined amounts of the immune cells and myelomacells in the culture medium and then mixing in PEG solution, prewarmedto about 37° C. During cell fusion, PEG, with an average molecularweight of about 1000-6000, for example, can normally be added at aconcentration of 30%-60% (w/v). Subsequently, the cell fusion agents andother agents not amenable to hybridoma growth are removed by therepeated sequential addition of a suitable culture medium, as indicatedabove, centrifugation, and the removal of the supernatant.

The hybridomas thus obtained can be selected with a selective culturemedium corresponding to the selection marker possessed by the myelomaused for cell fusion. For example, HGPRT- or TK-deficient cells can beselected by culture in HAT culture medium (culture medium containinghypoxanthine, aminopterin, and thymidine). When HAT-sensitive myelomacells are used for cell fusion, those cells that have successfully fusedwith normal cells can be selectively grown in HAT culture medium.Culture in HAT medium is continued for an adequate amount of time forcells other than the target hybridomas (nonfused cells) to die.Specifically, the target hybridomas can generally be selected by culturefor several days to several weeks. Next, screening and monocloning for ahybridoma that produces the target antibody can be performed with anordinary limiting dilution method. Alternatively, an antibody thatrecognizes AXL can be prepared using the method described inInternational Publication No. WO 03/104453.

Screening and monocloning for a target antibody is preferably carriedout with a known screening method based on an antigen-antibody reaction.For example, an antigen is bound to a carrier, such as polystyrene beadsor a commercially available 96-well microtiter plate, and reacted withthe culture supernatant of the hybridoma. The carrier is then washed,and reacted with an enzyme-labeled secondary antibody or the like. If atarget antibody that reacts with the sensitizing antigen is present inthe culture supernatant, the secondary antibody binds to the carrierthrough this antibody. Finally, whether or not the target antibody ispresent in the culture supernatant can be determined by detecting thesecondary antibody bound to the carrier. A hybridoma producing thedesired antibody, which can bind to the antigen, can be cloned by amethod such as limiting dilution. At this time, the antigen used forimmunization or a substantially equivalent AXL protein can be usedpreferentially as the antigen.

In addition to the method for producing a hybridoma by immunizing ananimal other than a human with an antigen, a target antibody can also beobtained by sensitizing human lymphocytes with the antigen.Specifically, human lymphocytes are first sensitized with AXL protein invitro. The immunosensitized lymphocytes are then fused to a suitablefusion partner. Myeloma cells of human origin, with the ability todivide continuously, for example, can be used as the fusion partner (seeJapanese Patent Application Kokoku Publication No. (JP-B) H1-59878(examined, approved Japanese patent application published foropposition)). An anti-AXL antibody produced with this method is a humanantibody with binding activity for AXL protein.

An anti-AXL human antibody can also be obtained by administering AXLprotein as the antigen to a transgenic animal with the entire repertoireof human antibody genes. Antibody-producing cells of the immunizedanimal can be immortalized by treatments such as fusion with a suitablefusion partner or infection with Epstein-Barr virus. An anti-AXLantibody can also be obtained by isolating a human antibody directedagainst AXL protein from immortalized cells obtained in this manner (seeInternational Publication Nos WO 94/25585, WO 93/12227, WO 92/03918, andWO 94/02602). Cells producing antibodies with target reactionspecificity can also be cloned by cloning the immortalized cells. Whenusing a transgenic animal as the immunized animal, the immune system ofthe animal recognizes human AXL as foreign. Thus, a human antibodydirected against human AXL can easily be obtained. A hybridoma producinga monoclonal antibody prepared in this manner can be subcultured inordinary culture medium. The hybridoma can also be stored for anextended period of time in liquid nitrogen.

The hybridoma can be cultured in accordance with ordinary methods toobtain the target monoclonal antibody from its culture supernatant.Alternatively, the monoclonal antibody can be produced by administeringthe hybridoma to a mammal compatible with it to allow the hybridoma togrow, using the resulting ascites as the monoclonal antibody. The formermethod is suitable for obtaining highly pure antibody.

In the present invention, an antibody encoded by antibody genes clonedfrom antibody-producing cells can also be used. Cloned antibody genescan be expressed as antibody by incorporating them in a suitable vectorand introducing the vector into a host. Methods for isolating theantibody genes, introducing them into a vector, and transforming hostcells with it have already been established (see, for example, Vandamme,A. M. et al., Eur. J. Biochem. (1990) 192, 767-775).

For example, a cDNA encoding a variable region (V region) of theanti-AXL antibody can be obtained from hybridoma cells producing theanti-AXL antibody. To accomplish this, total RNA is usually firstextracted from the hybridoma. Examples of methods for extracting mRNAfrom cells include guanidine ultracentrifugation (Chirgwin, J. M. etal., Biochemistry (1979) 18, 5294-5299) and the AGPC method(Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159).

The extracted mRNA can be purified using an mRNA Purification Kit (GEHealthcare Bio-sciences) and the like. Alternatively, kits such as theQuickPrep mRNA Purification Kit (GE Healthcare Bio-sciences) arecommercially available for the extraction of all mRNAs directly fromcells. These kits can be used to obtain all mRNAs from a hybridoma. ThecDNA encoding an antibody V region can be synthesized from the resultingmRNAs using reverse transcriptase. The cDNA can be synthesized with, forexample, the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit(Seikagaku Corp.). The 5′-Ampli FINDER RACE Kit (Clontech) and 5′-RACEmethod using PCR (Frohman, M. A. et al., Proc. Natl Acad. Sci. U.S.A(1988) 85, 8998-9002, Belyaysky, A. et al., Nucleic Acids Res. (1989)17, 2919-2932) can be used to synthesize and amplify the cDNA. Suitablerestriction sites, described below, can also be introduced at both endsof the cDNA during the course of cDNA synthesis.

Target cDNA fragments are purified from the resulting PCR product andlinked to vector DNA. A recombinant vector is thus prepared, and afterits introduction into Escherichia coli or the like and the selection ofcolonies, the desired recombinant vector can be prepared from the E.coli that formed colonies. Whether or not the recombinant vector has thenucleotide sequences of the target cDNA can be confirmed by a knownmethod, such as dideoxynucleotide chain termination sequencing.

PCR using a primer that amplifies a variable region gene can also beused to obtain a gene encoding a variable region. First, cDNA issynthesized using the extracted mRNA as the template to construct a cDNAlibrary. It is convenient to use a commercially available kit tosynthesize the cDNA library. Because the amount of mRNA obtained fromonly a small number of cells is extremely small, its direct purificationresults in a low yield. Thus, mRNA is normally purified after theaddition of a carrier RNA that clearly does not contain any antibodygene. Alternatively, when it is possible to extract a certain amount ofRNA, RNA from only antibody-producing cells can be efficientlyextracted. For example, the addition of carrier RNA may not be necessaryfor the extraction of RNA from 10 or more, 30 or more, or preferably 50or more antibody-producing cells.

The antibody gene is then amplified by PCR using the cDNA library thusconstructed as the template. Primers for amplifying the antibody genesby PCR are known. For example, primers to amplify human antibody genescan be designed based on the literature (for example, J. Mol. Biol.(1991) 222, 581-597). These primers have nucleotide sequences thatdiffer for each immunoglobulin subclass. Thus, when a cDNA library of anunknown subclass is used as the template, PCR is performed with allpossibilities considered.

For example, when a gene encoding human IgG is to be obtained, primersthat amplify a gene encoding γ1 to γ5 as heavy chains and κ and λ chainsas light chains can be used. To amplify a variable region gene of IgG, aprimer that anneals to a sequence corresponding to the hinge region istypically used for the primer on the 3′ side. Conversely, a primercorresponding to each subclass can be used for the primer on the 5′side.

The PCR products amplified with primers that amplify the genes of eachsubclass of heavy chains and light chains are made into independentlibraries. The use of a library synthesized in this manner makes itpossible to reconstitute immunoglobulins comprised of combinations ofheavy chains and light chains. A target antibody can then be screenedfor using the binding activity of the reconstituted immunoglobulins toAXL as an indicator.

After a cDNA encoding a V region of the target anti-AXL antibody isobtained, the cDNA is digested with a restriction enzyme that recognizesa restriction site inserted into both ends of the cDNA. A preferredrestriction enzyme recognizes and digests a nucleotide sequence that isunlikely to occur in the nucleotide sequence constituting the antibodygene. A restriction enzyme that imparts a cohesive end is preferablewhen inserting a single copy of the digested fragment into a vector inthe proper direction. An antibody expression vector can be generated byinserting the cDNA encoding V regions of the anti-AXL antibody, digestedas described above, into a suitable expression vector. At this time, achimeric antibody can be produced by fusing in frame genes encoding anantibody constant region (C region) and genes encoding the V regiondescribed above. Herein, “chimeric antibody” refers to an antibodycontaining constant and variable regions derived from differentorganisms. Thus, xenogeneic chimeric antibodies, such as mouse-humanantibodies and human-human allogeneic chimeric antibodies, are includedin the chimeric antibodies of the present invention. A chimeric antibodyexpression vector can also be constructed by inserting the V regiongenes into an expression vector that originally had constant regions.

Specifically, the recognition sequence of a restriction enzyme thatdigests the V region gene can be arranged on the 5′ side of anexpression vector retaining a DNA encoding the desired antibody constantregion (C region). A chimeric antibody expression vector is constructedby digesting the two with the same combination of restriction enzymesand then fusing them in frame.

Antibody genes can be incorporated into an expression vector forexpression under the control of an expression control domain to producethe anti-AXL antibody of the present invention. An expression controldomain for expressing antibody can include, for example, an enhancer anda promoter. Recombinant cells expressing DNA encoding the anti-AXLantibody can then be obtained by transforming suitable host cells withthis expression vector.

In the expression of antibody genes, DNAs encoding the antibody heavychain (H chain) and light chain (L chain) can each be incorporated intodifferent expression vectors. Vectors incorporating either the H chainor the L chain can express an antibody molecule with the H chain and Lchain after the vectors are simultaneously transformed (cotransfected)into the same host cell. Alternatively, DNAs encoding H chain and Lchain can be incorporated in a single expression vector to transformhost cells (see International Publication No. WO 94/11523).

Many combinations of hosts and expression vectors are known for thepreparation of antibodies by first isolating antibody genes and thenintroducing them into a suitable host. All of these expression systemscan be applied to the present invention. Animal cells, plant cells, orfungal cells can be used when eukaryotic cells are used as hosts.Specific examples of animal cells that can be used in the presentinvention include mammalian cells (such as CHO, COS, myeloma, BHK [babyhamster kidney], Hela, and Vero cells), amphibian cells (such as Xenopusoocytes), and insect cells (such as sf9, sf21, and Tn5 cells).

Known examples of plant cells used in antibody gene expression systemsare cells from the genus Nicotiana, such as Nicotiana tabacum.Callus-cultured cells can be used for plant cell transformation.

Examples of fungal cells that can be used include those of yeast (thegenus Saccharomyces, such as Saccharomyces cerevisiae, and themethanol-utilizing yeast genus Pichia, such as Pichia pastoris) and offilamentous fungi (the genus Aspergillus, such as Aspergillus niger).

Antibody gene expression systems that use prokaryotic cells are alsoknown. For example, cells of bacteria such as E. coli or Bacillussubtilis can be used in the present invention. When using mammaliancells, an expression vector can be constructed in which a routinely useduseful promoter, the antibody genes to be expressed, and a polyA signalat the 3′ side downstream from it are operably linked. An example of apromoter/enhancer is human cytomegalovirus immediate earlypromoter/enhancer.

Other examples of promoter/enhancers that can be used to express anantibody of the present invention include viral promoter/enhancers ormammalian cell promoter/enhancers, such as human elongation factor 1α(HEF1α). Specific examples of viruses whose promoter/enhancers areuseful include retroviruses, polyomaviruses, adenoviruses, and simianvirus 40 (SV40).

When using an SV40 promoter/enhancer, the method of Mulligan et al. canbe used (Nature (1979) 277, 108). An HEF1α promoter/enhancer can also beused to easily express a target gene with the method of Mizushima et al.(Nucleic Acids Res. (1990) 18, 5322).

With E. coli, antibody genes can be expressed by operably linking aroutinely used useful promoter, an antibody secretion signal sequence,and the antibody genes to be expressed. Examples of promoters includethe lacZ promoter and the araB promoter. When using the lacZ promoter,the method of Ward et al. can be used (Nature (1989) 341, 544-546; FASEBJ. (1992) 6, 2422-2427). Alternatively, the araB promoter can be used toexpress target genes according to the method of Better et al. (Science(1988) 240, 1041-1043).

An example of the antibody secretion signal sequence that can be usedfor the production into the periplasm of E. coli is the pelB signalsequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379). Theantibody produced in the periplasm is separated and then structurallyrefolded using a protein denaturant such as a guanidine hydrochloride orurea so that the antibody has the desired binding activity.

Examples of useful replication origins that can be inserted into anexpression vector include those originating in SV40, polyomaviruses,adenoviruses, and bovine papillomavirus (BPV). A selection marker canalso be inserted into the expression vector to amplify the number ofgene copies in a host cell system. Specific examples of selectionmarkers that can be used include the aminoglycoside transferase (APH)gene, the thymidine kinase (TK) gene, the E. coli xanthine-guaninephosphoribosyl transferase (Ecogpt) gene, and the dihydrofolatereductase (dhfr) gene.

A target antibody is produced by introducing these expression vectorsinto host cells and culturing the transformed host cells in vitro or invivo. Culture of the host cells is carried out in accordance with knownmethods. Examples of culture media that can be used include DMEM, MEM,RPMI1640, and IMDM, and these can be used in combination with a serumsupplement such as FCS.

An antibody expressed and produced in the manner described above can bepurified using known methods that are routinely used for proteinpurification, either alone or in a suitable combination. For example,antibodies can be separated and purified by the suitable selection andcombination of, for example, an affinity column such as a Protein Acolumn, a chromatography column, a filter, ultrafiltration, salting out,or dialysis (Antibodies—A Laboratory Manual, Ed Harlow and David Lane,Cold Spring Harbor Laboratory, 1988).

In addition to the host cells described above, a transgenic animal canalso be used to produce the recombinant antibody. A target antibody canbe obtained from an animal into which a gene encoding the targetantibody has been introduced. For example, antibody genes can beconstructed as fused genes by inserting them into a gene encoding aprotein inherently produced in frame in milk. Goat β casein, forexample, can be used as this protein secreted in milk. A DNA fragmentcontaining the fused genes into which the antibody genes have beeninserted is injected into a goat embryo and the injected embryo isintroduced into a female goat. The desired antibody can be acquired inthe form of a fusion protein, fused to milk protein, from milk producedby the transgenic goat (or offspring thereof) born from the goat thatreceived the embryo. Hormones can be given as appropriate to thetransgenic goat to increase the amount of milk containing the desiredantibody produced by it (Ebert, K. M. et al., Bio/Technology (1994) 12,699-702). A C region originating in an animal antibody can be used forthe C region of a recombinant antibody of the present invention.Examples of useful mouse antibody H chain C regions include Cγ1, Cγ2a,Cγ2b, Cγ3, Cδ, Cα1, Cα2, and Cε, and examples of L chain C regionsinclude Cκ and Cλ. Examples of useful animal antibodies other than mouseantibodies include rat, rabbit, goat, sheep, camel, and monkeyantibodies. The sequences of these antibodies are known. The C regioncan also be modified to improve the stability of the antibody or itsproduction. In the present invention, when administering the antibody toa human, an artificially modified recombinant antibody can be made inorder to, for example, lower its xenogeneic antigenicity in humans.Examples of recombinant antibodies include chimeric antibodies andhumanized antibodies.

These modified antibodies can be produced using known methods. Chimericantibodies refer to antibodies in which variable regions and constantregions of different origins are linked. For example, an antibody withheavy chain and light chain variable regions of a mouse antibody andheavy chain and light chain constant regions of a human antibody is amouse-human xenogeneic chimeric antibody. A recombinant vectorexpressing a chimeric antibody can be prepared by linking DNA encodingvariable regions of a mouse antibody with a DNA encoding a constantregion of a human antibody and then incorporating it into an expressionvector. Recombinant cells transformed with the vector are cultured andthe incorporated DNAs are expressed to obtain the chimeric antibodyproduced in a culture. C regions of a human antibody are used as the Cregions of chimeric antibodies and humanized antibodies. For example,Cγ1, Cγ2, Cγ3, Cγ4, Cδ, Cα1, Cα2, and Cε can be used for the C region inH chains. Cκ and Cλ, can be used for the C region in L chains. The aminoacid sequences of these C regions and the nucleotide sequences thatencode them are known. A human antibody C region can also be modified toimprove the stability of the antibody itself or the antibody production.

In general, chimeric antibodies are composed of V regions originatingfrom antibodies of an animal other than a human and C regionsoriginating from human antibodies. In contrast, humanized antibodies arecomposed of complementarity determining regions (CDRs) originating fromantibodies of animals other than humans, framework regions (FRs)originating from human antibodies, and C regions originating from humanantibodies. Because humanized antibodies have reduced antigenicity inthe human body, they are useful as an active ingredient of a therapeuticagent of the present invention.

Antibody variable regions are normally composed of three CDRs flanked byfour FRs. A CDR is substantially a region that determines the bindingspecificity of an antibody. The amino acid sequences of CDRs are rich indiversity. Conversely, the amino acid sequences that constitute FRsoften demonstrate high homology, even among antibodies with differentbinding specificities. Consequently, it is generally considered that thebinding specificity of a certain antibody can be grafted onto anotherantibody by grafting the CDRs.

A humanized antibody is also referred to as a “reshaped” human antibody.Specifically, humanized antibodies in which the antibody CDRs of ananimal other than a human, such as a mouse, have been grafted onto humanantibodies, are known. General genetic recombination techniques forproducing humanized antibodies are also known.

A specific example of a known method of grafting the CDRs of a mouseantibody to human FRs is overlap extension PCR. In the overlap extensionPCR, a nucleotide sequence encoding a CDR of the mouse antibody to begrafted is added to primers used to synthesize a human antibody FR.Primers are prepared for each of the four FRs. In general, it isconsidered to be advantageous in terms of maintaining the CDR functionto select a human FR with high homology to the mouse FR when grafting amouse CDR onto a human FR. That is, it is generally preferable to use ahuman FR with an amino acid sequence with high homology to the aminoacid sequence of the FR adjacent to the mouse CDR to be grafted.

The nucleotide sequences to be linked are designed so that they aremutually connected in frame. Human FRs are individually synthesized byspecific primer sets. As a result, products are obtained in which a DNAthat encodes a mouse CDR has been added to each FR. The nucleotidesequences encoding mouse CDRs of the products are designed to overlapone another. A complementary-strand synthesis reaction is then carriedout by mutually annealing the overlapping CDR portions of the productssynthesized using the human antibody gene as the template. As a resultof this reaction, human FRs are linked through the mouse CDR sequence.

Finally, the full length of a V region gene in which three CDRs and fourFRs have been linked is amplified by primers that anneal to its 5′ and3′ ends and which have suitable restriction enzyme recognition sequencesadded. A vector for expressing the humanized antibody can then beprepared by inserting the DNA obtained in the manner described above andDNA encoding a human antibody C region into an expression vector so thatthey are fused in frame. The humanized antibody is then produced in aculture of cultured cells by introducing the recombinant vector into ahost to establish recombinant cells, followed by culturing therecombinant cells and expressing the DNA encoding the humanized antibody(see European Patent Publication No. EP 239400 and InternationalPublication No. WO 96/02576).

FRs of a human antibody can be preferentially selected so that the CDRsform a favorable antigen-binding site when linked through the CDRs, byqualitatively or quantitatively measuring and evaluating its bindingactivity to the antigen of the humanized antibody prepared in the mannerdescribed above. Amino acid residues of the FRs can also be substitutedas necessary, so that the CDRs of the reshaped human antibody form asuitable antigen-binding site. For example, an amino acid sequencemutation can be introduced into FRs by applying PCR used to graft themouse CDRs to the human FRs. Specifically, a mutation of a partialnucleotide sequence can be introduced into a primer that anneals to theFR. A mutated nucleotide sequence is introduced into the FR synthesizedwith such a primer. A mutant FR sequence with a desired property can beselected by measuring and evaluating the binding activity of theamino-acid-substituted mutant antibody to the antigen, using the methoddescribed above (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

Methods for acquiring human antibodies are also known. For example,human lymphocytes are sensitized with the desired antigen or cellsexpressing the desired antigen in vitro. Next, the desired humanantibody with binding activity for the antigen can be acquired by fusingthe sensitized lymphocytes to human myeloma cells (see JP-B H1-59878).U266 cells, for example, can be used as the human myeloma cells, toserve as the fusion partner.

A desired human antibody can also be acquired by immunizing with thedesired antigen a transgenic animal with the entire repertoire of humanantibody genes (see International Publication Nos WO 93/12227, WO92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).Moreover, technologies by which human antibodies can be acquired bypanning, using a human antibody library, are also known. For example,the V region of a human antibody can be expressed on the surface of aphage in the form of a single-chain antibody (scFv) using the phagedisplay method, thus allowing the selection of a phage that binds to anantigen. By analyzing the genes of the selected phage, it is possible todetermine the DNA sequence encoding the V region of the human antibodythat binds to the antigen. After determining the DNA sequence of thescFv that binds to the antigen, the V region sequence is fused in frameto the sequence of the C region of the desired human antibody, and isthen inserted into a suitable expression vector to prepare an expressionvector. The human antibody can be acquired by introducing the expressionvector into the preferred expression cells, as described above, andexpressing the gene encoding the human antibody. These methods arealready known (International Publication Nos WO 92/01047, WO 92/20791,WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388).

The antibodies of the present invention include not only bivalentantibodies as represented by IgG, but also monovalent antibodies orpolyvalent antibodies as represented by IgM, as long as they bind to theAXL protein. The polyvalent antibodies of the present invention includethose with the same antigen-binding sites, and those in which some orall of the antigen-binding sites are different. The antibody of thepresent invention is not limited to the entire antibody molecule, butmay also be a minibody or modified antibody thereof, as long as it bindsto the AXL protein.

Minibodies include antibody fragments in which a portion of the wholeantibody (such as whole IgG) is deleted. Partial deficiencies inantibody molecules are permitted as long as the ability to bind to theAXL antigen is retained. The antibody fragment of the present inventionpreferably comprises one or both of the heavy chain variable regions(VH) and light chain variable regions (VL). The amino acid sequences ofVH or VL can comprise substitutions, deletions, additions, and/orinsertions. Moreover, a portion of one or both of VH and VL can bedeleted as long as the ability to bind to the AXL antigen is retained.The variable regions may also be chimerized or humanized. Specificexamples of antibody fragments include, for example, Fab, Fab′, F(ab′)2,and Fv. Specific examples of minibodies include Fab, Fab′, F(ab′)2, Fv,scFV (single-chain Fv), diabody, sc(Fv)2 (single-chain (Fv)2), etc.Polymers of these antibodies (such as dimers, trimers, tetramers, orpolymers) are also included in the minibodies of the present invention.

Antibody fragments can be obtained by producing an antibody fragment bytreating the antibody with an enzyme. Known examples of enzymes used toproduce antibody fragments include papain, pepsin, plasmin, etc.Alternatively, genes encoding these antibody fragments can beconstructed, introduced into an expression vector, and then expressed insuitable host cells (see, for example, Co, M. S. et al., J. Immunol.(1994) 152, 2698-2976; Better, M. and Horwitz, A. H., Methods inEnzymology (1989) 178, 476-496; Plueckthun, A. and Skerra, A., Methodsin Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology(1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989)121, 663-669; and Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

Digestive enzymes cleave a specific position of an antibody fragment toyield an antibody fragment with a specific structure, as indicatedbelow. An arbitrary portion of an antibody can be deleted by applyinggenetic engineering techniques to an antibody fragment enzymaticallyobtained in this manner.

Papain digestion: F(ab)2 or Fab

Pepsin digestion: F(ab′)2 or Fab′

Plasmin digestion: Facb

“Diabody” refers to bivalent antibody fragments constructed by genefusion (see Holliger, P. et al., Proc. Natl Acad. Sci. U.S.A (1993) 90,6444-6448; EP 404,097; WO 93/11161, etc.). Diabodies are dimers composedof two polypeptide chains. Normally, VL and VH within the same chain ofthe polypeptide chains that forms a dimer are both bound by linkers. Thelinkers in a diabody are typically too short to allow the VL and VH tobind to each other. Specifically, the number of amino acid residues thatconstitute a linker is, for example, about five residues. Thus, the VLand VH encoded in the same polypeptide chain cannot form a single-chainvariable region fragment, but instead form a dimer with a differentsingle-chain variable region fragment. As a result, a diabody has twoantigen-binding sites.

An scFv is obtained by linking the H chain V region and the L chain Vregion of an antibody. In an scFv, the H chain V region and L chain Vregion are linked through a linker, preferably a peptide linker (Huston,J. S. et al., Proc. Natl Acad. Sci. U.S.A (1988) 85, 5879-5883). The Hchain V region and L chain V region in an scFv may be derived from anyantibody described as an antibody herein. There is no particularlimitation on the peptide linkers that link the V regions. For example,any arbitrary single-chain peptide comprising about three to 25 residuescan be used as a linker. The V regions can be linked by, for example,the PCR method described above. To link the V regions using the PCRmethod, a DNA encoding the entire or desired partial amino acid sequenceof the DNA sequence encoding the H chain or the H chain V region of theabove antibody, and a DNA sequence encoding the L chain or the L chain Vregion of the above antibody, are used as templates.

DNA encoding the V regions of the H chain and that encoding L chain areboth amplified by the PCR method using pairs of primers with sequencescorresponding to the sequences at both ends of the DNA to be amplified.Next, DNA encoding the peptide linker portion is prepared. The DNAencoding the peptide linker can also be synthesized by PCR. Nucleotidesequences that can link the amplification products of each separatelysynthesized V region are added to the 5′ side of the primers used atthis time. Next, a PCR reaction is carried out using the “H chain Vregion DNA”, the “peptide linker DNA”, and the “L chain V region DNA”together with the primers for the assembly PCR. The primers for theassembly PCR consist of a combination of a primer that anneals to the 5′side of the “H chain V region DNA” and a primer that anneals to the 3′side of the “L chain V region DNA”. Therefore, the primers for theassembly PCR consist of a primer set that can amplify the DNA encodingthe entire sequence of the scFv to be synthesized. Conversely,nucleotide sequences that can link to each V region DNA are added to the“peptide linker DNA”. As a result, these DNAs are linked together andthe full length of scFv is finally produced as an amplification productof the primers used for the assembly PCR. Once a DNA encoding an scFv isprepared, an expression vector comprising the DNA and recombinant cellstransformed with the expression vector can be acquired with ordinarymethods. The scFv can also be acquired by expressing the DNA encodingthe scFv in cultures of the resulting recombinant cells.

An sc(Fv)2 is a minibody in which two VHs and two VLs are linked by alinker or such to form a single chain (Hudson, et al., J. Immunol.Methods (1999) 231, 177-189). An sc(Fv)2 can be prepared, for example,by connecting scFvs with a linker.

An sc(Fv)2 is preferably an antibody in which two VHs and two VLs arearranged in the order VH, VL, VH, VL (VH-linker-VL-linker-VH-linker-VL)using the N-terminal side of a single-chain polypeptide as the startingpoint.

Any arbitrary peptide linker that can be introduced by geneticengineering, a synthetic compound linker (for example, those disclosedin Protein Engineering, (1996) 9(3), 299-305) or such, can be used asthe linker to link antibody variable regions. Peptide linkers arepreferred in the present invention. There is no particular limitation onthe length of the peptide linkers, and the length can be suitablyselected by those skilled in the art according to the purpose of use.Normally, the number of amino acid residues constituting a peptidelinker ranges from one to 100 amino acids, preferably from three to 50amino acids, more preferably from five to 30 amino acids, andparticularly preferably from 12 to 18 amino acids (for example, 15 aminoacids).

The amino acid sequence constituting a peptide linker can be anyarbitrary sequence as long as it does not inhibit the binding functionof the scFv.

Alternatively, V regions can be linked using a synthetic chemical linker(chemical cross-linking agent). Cross-linking agents ordinarily used tocross-link peptide compounds and such can be used in the presentinvention. Examples of cross-linking agents that can be used includeN-hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS),bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate)(DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycolbis(succinimidylsuccinate) (EGS), ethyleneglycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate(DST), disulfosuccinimidyl tartrate (sulfo-DST),bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), andbis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

Normally, three linkers are required when four antibody variable regionsare linked. The multiple linkers used may be identical or different. Apreferred minibody of the present invention is a diabody or sc(Fv)2. Toobtain these minibodies, an antibody is treated with an enzyme such aspapain or pepsin to produce antibody fragments. Alternatively, a DNAencoding these antibody fragments is constructed, introduced into anexpression vector, and then expressed in suitable host cells (see, forexample, Co, M. S. et al., J. Immunol. (1994) 152, 2698-2976; Better, M.and Horwitz, A. H., Methods Enzymol. (1989) 178, 476-496; Plueckthun, A.and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E.,Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., MethodsEnzymol. (1986) 121, 663-669; and Bird, R. E. and Walker, B. W., TrendsBiotechnol. (1991) 9, 132-137).

The antibody of the present invention can also be used as a modifiedantibody bound to various molecules such as polyethylene glycol (PEG) orcytotoxic substances. Such modified antibodies can be obtained bychemically modifying an antibody of the present invention. Antibodymodification methods have already been established in the art.

The antibody of the present invention may also be a bispecific antibody.“Bispecific antibody” refers to an antibody that has variable regionsthat recognize different epitopes within the same antibody molecule. Theepitopes may be present in different molecules or present in the samemolecule. In the present invention, a bispecific antibody can haveantigen-binding sites that recognize different epitopes on an AXLmolecule. Alternatively, a bispecific antibody can recognize AXL via onerecognition site and a cytotoxic substance by the other recognitionsite. These antibodies are also included in the antibodies of thepresent invention.

A bispecific antibody that recognizes an antigen other than AXL can becombined in the present invention. For example, a bispecific antibodythat recognizes an antigen other than AXL, which is specificallyexpressed on the surfaces of target cancer cells in the same manner asAXL, can be combined.

Methods for producing bispecific antibodies are known. For example, abispecific antibody can be produced by linking two types of antibodiesthat recognize different antigens. Each of the linked antibodies may bea half molecule, with the H and L chains, or a quarter moleculecomprising only the H chain. Alternatively, fused cells that producebispecific antibodies can be prepared by fusing hybridomas producingdifferent monoclonal antibodies. Bispecific antibodies can also beprepared with genetic engineering techniques.

Binding Activity of an Antibody

Known means can be used to measure the antigen-binding activity of anantibody (Antibodies A Laboratory Manual, Ed Harlow and David Lane, ColdSpring Harbor Laboratory, 1988). Examples of methods that can be usedinclude ELISA (enzyme-linked immunosorbent assay), EIA (enzymeimmunoassay), RIA (radioimmunoassay), fluorescent immunoassay, etc.Examples of the means for measuring the binding activity of an antibodyto an antigen expressed in cells include the method described on pages359-420 of “Antibodies A Laboratory Manual” mentioned above.

Methods using a flow cytometer are particularly preferably used tomeasure the binding between an antigen expressed on the surface of cellssuspended in a buffer and such, and an antibody to that antigen.Examples of flow cytometers used include the FACSCanto™ II, FACSAria™,FACSArray™, FACSVantage™ SE, and FACSCalibur™ (all from BD Biosciences),and the EPICS ALTRA HyPerSort, Cytomics FC 500, EPICS XL-MCL ADC, EPICSXL ADC, and Cell Lab Quanta/Cell Lab Quanta SC (all from BeckmanCoulter).

An example of a preferred method of measuring the binding activity of atest AXL antibody to an antigen is the method of staining with asecondary antibody labeled with FITC, which recognizes a test antibodyreacted with cells expressing AXL, and then measuring with theFACSCalibur (BD Biosciences) and analyzing the fluorescence intensityusing CellQuest software (BD Biosciences).

Hybridomas

The present invention also provides hybridomas deposited under AccessionNos. FERM BP-10858 (AX285), FERM BP-10859 (AX292), FERM BP-10853(AX223), FERM BP-10852 (AX96), FERM BP-10856 (AX258), FERM BP-10857(AX284), FERM BP-10850 (Ax7), FERM BP-10851 (Ax51), FERM BP-10854(Ax225), and FERM BP-10855 (Ax232). These hybridomas produce anti-AXLantibodies with agonistic activity, anti-AXL antibodies withantagonistic activity, anti-AXL antibodies with activity that lowers theexpression level of AXL, anti-AXL antibodies with angiogenesisinhibitory activity, and/or anti-AXL antibodies withcell-growth-suppressing activity.

Angiogenesis Inhibitors

The present invention also provides angiogenesis inhibitors comprisingan anti-AXL antibody. There is no particular limitation on the mechanismby which angiogenesis is inhibited. Examples of the mechanism include aninhibitory effect on the migration activity of vascular endothelialcells, an apoptosis-inducing action on vascular endothelial cells, andan inhibitory effect on the vascular morphogenesis of vascularendothelial cells. The angiogenesis inhibitors of the present inventionpreferably inhibit angiogenesis in cancer tissues. There is noparticular limitation on the cancer tissues. Examples of these cancertissues include pancreatic cancer tissues (pancreatic adenocarcinomatissues, etc.), gastric cancer tissues, lung cancer tissues (tissues ofsmall-cell lung cancer, non-small-cell lung cancer, and such),osteosarcoma tissues, colon cancer tissues, prostate cancer tissues,melanoma tissues, endometrial cancer tissues, ovarian cancer tissues,uterine leiomyosarcoma tissues, thyroid cancer tissues, cancer stem celltissues, breast cancer tissues, bladder cancer tissues, renal cancertissues, glioma tissues, neuroblastoma tissues, and esophageal cancertissues. More preferable tissues are glioma tissue, gastric cancertissue, endometrial cancer tissue, non-small-cell lung cancer tissue,pancreatic adenocarcinoma tissue, and breast cancer tissue, particularlypancreatic adenocarcinoma tissue and breast cancer tissue.

There is no particular limitation on the antibodies used in theangiogenesis inhibitors of the present invention, as long as they havean angiogenesis inhibitory effect. For example, the antibodies describedabove (antibodies with agonistic activity, antibodies with antagonisticactivity, antibodies with activity that lowers the expression level ofAXL, etc.) can be used.

The angiogenesis inhibitors comprising the anti-AXL antibody of thepresent invention can be expressed as methods for inhibitingangiogenesis using an anti-AXL antibody. The angiogenesis inhibitorscomprising the anti-AXL antibody of the present invention can beexpressed as use of an anti-AXL antibody for producing an angiogenesisinhibitor.

Cell-Growth Suppressants

The present invention also provides cell-growth suppressants comprisinganti-AXL antibodies. There is no particular limitation on the mechanismby which cell growth is suppressed. Examples of the mechanisms includethose based on the angiogenesis inhibitory action, those based on thecytotoxic activity of the antibody, and those based on a cytotoxicsubstance bound to the antibody, but those based on the angiogenesisinhibitory action are preferable.

There is no particular limitation on the cells whose growth issuppressed by an anti-AXL antibody. The cells are preferably thoserelated to a disease, and more preferably cancer cells. Thus, examplesof the preferred embodiments of the cell-growth suppressants of thepresent invention include an anticancer agent comprising an anti-AXLantibody. When the cells are cancer cells, there is no particularlimitation on the type of cancer, and the types include pancreaticcancer (pancreatic adenocarcinoma, etc.), gastric cancer, lung cancer(small-cell lung cancer, non-small-cell lung cancer, and such),osteosarcoma, colon cancer, prostate cancer, melanoma, endometrialcancer, ovarian cancer, uterine leiomyosarcoma, thyroid cancer, cancerstem cell, breast cancer, bladder cancer, renal cancer, glioma,neuroblastoma, and esophageal cancer. More preferable cancers areglioma, gastric cancer, endometrial cancer, non-small-cell lung cancer,pancreatic adenocarcinoma, and breast cancer, particularly pancreaticadenocarcinoma and breast cancer.

There is no particular limitation on the antibodies used in thecell-growth suppressants of the present invention, as long as they havecell-growth-suppressing activity. For example, the antibodies describedabove (antibodies with agonistic activity, antibodies with antagonisticactivity, antibodies with activity that lowers the expression level ofAXL, etc.) can be used.

The cell-growth suppressants comprising the anti-AXL antibody of thepresent invention can be expressed as methods for suppressing cellgrowth using an anti-AXL antibody. When the cells whose growth issuppressed are cancer cells, the anticancer agents comprising theanti-AXL antibody of the present invention can be expressed as methodsfor treating and/or preventing cancer using an anti-AXL antibody. Thecell-growth suppressants comprising the anti-AXL antibody of the presentinvention can be expressed as use of an anti-AXL antibody to produce acell-growth suppressant. When the cells whose growth is suppressed arecancer cells, they can be expressed as use of an anti-AXL antibody forproducing an anticancer agent.

Phosphorylation Inducers

The present invention also provides phosphorylation inducers comprisingan anti-AXL antibody. The phosphorylation inducers of the presentinvention normally induce phosphorylation in cells expressing AXL.Although there is no particular limitation on the targets of thephosphorylation induction, the targets are normally polypeptides havingtyrosine and are preferably AXL.

There is no particular limitation on the antibodies used in thephosphorylation inducers of the present invention. For example, theantibodies with agonistic activity described above can be used.

The phosphorylation inducers comprising the anti-AXL antibody of thepresent invention can be expressed as methods for inducingphosphorylation using an anti-AXL antibody. The phosphorylation inducerscomprising the anti-AXL antibody of the present invention can also beexpressed as use of an anti-AXL antibody for producing a phosphorylationinducer.

Phosphorylation Inhibitors

The present invention also provides phosphorylation inhibitorscomprising an anti-AXL antibody. The phosphorylation inhibitors of thepresent invention normally inhibit the phosphorylation induced by thebinding of an AXL ligand (such as Gash) to AXL. Although there is noparticular limitation on the targets of phosphorylation inhibition, thetargets are normally polypeptides having tyrosine and are preferablyAXL.

There is no particular limitation on the antibodies used in thephosphorylation inhibitors of the present invention. For example, theantibodies with antagonistic activity described above can be used.

The phosphorylation inhibitors comprising the anti-AXL antibody of thepresent invention can be expressed as methods for inhibitingphosphorylation using an anti-AXL antibody. The phosphorylationinhibitors comprising the anti-AXL antibody of the present invention canalso be expressed as use of an anti-AXL antibody for producing aphosphorylation inhibitor.

Agents for Lowering the AXL Expression Level

The present invention also provides agents that lower the AXL expressionlevel comprising an anti-AXL antibody. The agent that lowers the AXLexpression level reduces AXL expression level in cells expressing AXL.There is no particular limitation on the cells that express AXL.Examples of these cells include cancer cells (Calu-1, MDA-MB-231,DU-145, etc.).

The reduction in the expression level of AXL may be a reduction in theamount of AXL already present by the degradation of AXL, or such, or maybe a reduction in the amount of newly expressed AXL by suppressing theexpression of AXL.

The agents that lower the AXL expression level comprising the anti-AXLantibody of the present invention can be expressed as methods forlowering the expression level of AXL using an anti-AXL antibody.Moreover, the agents that lower the AXL expression level comprising theanti-AXL antibody of the present invention can be expressed as use of ananti-AXL antibody for producing an agent for lowering the AXL expressionlevel.

Pharmaceutical Compositions

The angiogenesis inhibitors, cell-growth suppressants, phosphorylationinducers, phosphorylation inhibitors, or agents that lower the AXLexpression level of the present invention can be administered by eitheroral administration methods or parenteral administration methods.Parenteral administration methods are particularly preferred. Specificexamples of such administration methods include injectionadministration, transnasal administration, transpulmonaryadministration, and transcutaneous administration. The pharmaceuticalcompositions of the present invention can be administered systemicallyor locally by injection administration, for example, by intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, or such. Suitable methods of administration canalso be selected according to the age and symptoms of the patient. Thedosage can be selected, for example, within the range of 0.0001 mg to1000 mg per kilogram body weight per administration. Alternatively, thedosage can be selected, for example, within the range of 0.001 to100,000 mg/body per patient. However, the dosage of the pharmaceuticalcompositions of the present invention is not limited thereto.

The angiogenesis inhibitors, cell-growth suppressants, phosphorylationinducers, phosphorylation inhibitors, or agents for lowering the AXLexpression level of the present invention can be formulated according toordinary methods (for example, Remington's Pharmaceutical Science,latest edition, Mark Publishing Company, Easton, USA), and may comprisepharmaceutically acceptable carriers or additives. Examples of thecarriers and additives include, but are not limited to, surfactants,vehicles, colorants, fragrances, preservatives, stabilizers, buffers,suspension agents, isotonic agents, binders, disintegration agents,lubricants, fluidity promoters, and flavoring agents. Other commonlyused carriers can be used as appropriate. Specific examples of suchcarriers include light silicic anhydride, lactose, crystallinecellulose, mannitol, starch, carmellose calcium, carmellose sodium,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chainfatty-acid triglycerides, polyoxyethylene hydrogenated castor oil 60,saccharose, carboxymethyl cellulose, cornstarch, inorganic salts, etc.

All prior art reference cited herein are incorporated by reference intheir entirety.

EXAMPLES

Although the present invention will be explained in more detail by thefollowing Examples, the present invention is not limited by theseExamples.

Example 1 1-1 Preparation of Antigen

Hamster ovary cells (CHO (dhfr⁻) cells) were transfected with theexpression vector for a fusion protein (hAXL-ECD-mIgG2aFc), in which theextracellular domain of human AXL and an Fc domain of mouse IgG2a werefused, and CHO cell lines that produce hAXL-ECD-mIgG2aFc protein werecloned with G418 selection. The culture supernatant of thehAXL-ECD-mIgG2aFc protein-producing CHO cell lines collected usingserum-free medium (CHO-S-SFM II; Gibco) was added to a Protein G Column(HiTrap Protein G HP, GE Healthcare) equilibrated with a binding buffer(20 mM phosphate buffer, pH 7.0). After the unbound proteins were washedwith the binding buffer, fractions of hAXL-ECD-mIgG2aFc protein werecollected with an elution buffer (100 mM glycine-HCl, pH 2.7) into tubescontaining neutralizing buffer (1 M Tris-HCl, pH 9.0). Then the bufferof the purified protein was replaced with phosphate-bufferedphysiological saline (pH 7.35-7.65; Takara Bio) and the purified proteinwas concentrated using an ultrafiltration kit for a molecular weightfraction of 10 kDa (Centricon (registered trademark), Millipore). Theconcentration of the purified protein was calculated from the absorbanceat 280 nm using a molar absorption coefficient calculated according tothe calculation formula of Pace et al. (Prof Sci. (1995) 4, 2411-2423).

1-2 Preparation of Anti-AXL-Antibody-Producing Hybridoma

Four BALB/c mice (male, six weeks old at the start of immunization,Charles River Laboratories Japan) and two MRL/lpr mice (male, six weeksold at the start of immunization, Charles River Laboratories Japan) wereimmunized as described below with the antigen prepared in the previoussection (hAXL-ECD-mIgG2aFc protein). Antigen emulsified with Freund'scomplete adjuvant (H37 Ra, Difco Laboratories) was administeredsubcutaneously at 40 μg/head as the initial immunization. Two weekslater, antigen emulsified with Freund's incomplete adjuvant (DifcoLaboratories) was administered subcutaneously at 40 μg/head. The animalswere subsequently immunized three times more at one week intervals.Increases in the serum antibody titer in response to the antigen wereconfirmed by ELISA as indicated in the following section, followed by afinal immunization of intravenous administration of antigen diluted withphosphate-buffered physiological saline (phosphate-buffered salinewithout calcium ions or magnesium ions, PBS(−); Nissui Pharmaceutical)at 10 μg/head. Three days after the final immunization, mouse spleencells and mouse myeloma cells P3X63Ag8U.1 (referred to as P3U1, ATCCCRL-1597) were fused according to ordinary methods using PEG 1500 (RocheDiagnostics). The fused cells were cultured in RPMI1640 medium(Invitrogen) containing 10% FBS (Invitrogen) (hereafter referred to as10% FBS/RPMI1640). On the day after fusion, the fused cells weresuspended in semifluid medium (StemCells) followed by the selectiveculture and colonization of the hybridomas. Hybridoma colonies werepicked from the medium on the ninth or tenth day after fusion and seededinto a 96-well plate containing HAT selective medium (10% FBS/RPMI1640,2 vol % HAT 50× concentrate [Dainippon Pharmaceutical] and 5 vol %BM-Condimed H1 [Roche Diagnostics]) at one colony per well. Afterculture for three to four days, the supernatant was collected from eachwell and the hybridomas with binding activity to the extracellulardomain of human AXL were selected by measuring their binding activity tothe aforementioned antigen and to a control protein fused with the Fcdomain of mouse IgG2a by ELISA, as indicated in the following section.

The binding activities of the supernatants of the selected hybridomasare shown in Table 1.

TABLE 1 2nd AXL 2nd SC Abs 2nd SC Abs SC Abs 2nd SC Abs IgG Clone No.AXL-mFc FGFR2-mFc AbsΔ AXL-His Binding 7 2.053 0.057 1.996 1.118 0.66 511.844 0.058 1.786 0.538 0.55 232 1.353 0.061 1.292 1.204 0.575 96 2.1220.058 2.064 1.554 0.635 119 2.208 0.063 2.145 1.527 0.668 223 2.0760.071 2.005 1.542 0.339 225 0.629 0.055 0.574 0.642 0.859 258 2.0050.078 1.927 1.028 0.74 284 0.619 0.064 0.555 0.124 0.857 285 1.804 0.0581.746 0.914 0.965 292 1.877 0.069 1.808 1.234 1.052

The hybridomas selected by the present inventors were deposited at theInternational Patent Organism Depositary of the National Institute ofAdvanced Industrial Science and Technology. The following sectionprovides a description of the contents, specifying the deposition.

(a) Name and Address of the Depositary Institution

Name: International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology

Address: Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan305-8566

(b) Acceptance Date (Deposition Date): Jul. 5, 2007 (c) Accession No.

AXL No. 7 #070402 (Ax7) (Accession No. FERM BP-10850)

AXL No. 51 #070406 (Ax51) (Accession No. FERM BP-10851)

AXL No. 232 #070406 (Ax232) (Accession No. FERM BP-10855)

AXL No. 96 #070402 (Ax96) (Accession No. FERM BP-10852)

AXL No. 223 #070402 (Ax223) (Accession No. FERM BP-10853)

AXL No. 225 #070402 (Ax225) (Accession No. FERM BP-10854)

AXL No. 258 #070402 (Ax258) (Accession No. FERM BP-10856)

AXL No. 284 #070402 (Ax284) (Accession No. FERM BP-10857)

AXL No. 285 #070402 (Ax285) (Accession No. FERM BP-10858)

AXL No. 292 #070411 (Ax292) (Accession No. FERM BP-10859)

1-3 Binding Activity to Human AXL

Antigen (hAXL-ECD-mIgG2aFc) diluted to 1 μg/mL with coating buffer (100mM sodium bicarbonate [pH 9.6], 0.02% sodium azide) or control proteinfused with the Fc domain of mouse IgG2a was dispensed into a 96-wellplate (Nunc-Immuno™ 96 MicroWell™ MaxiSorp™ plate; Nalge NuncInternational) at 80 μL/well, followed by incubation at least overnightat 4° C. After it was washed three times with phosphate-buffered salinecontaining 0.05 vol % Tween (registered trademark) 20 (tPBS[−]), theplate was blocked at least overnight at 4° C. with diluent buffer (1/5dilution of BlockingOne; Nacalai Tesque). After the removal of thebuffer, mouse antiserum or hybridoma culture supernatant diluted withdiluent buffer was added to the plate at 80 μL/well, followed byincubation for one hour at room temperature. After the plate had beenwashed three times with tPBS(−), HRP-labeled anti-mouse IgG antibody(Stressgen), diluted 1/5000 with diluent buffer, was added at 80μL/well, followed by incubation for one hour at room temperature. Afterthe plate had been washed five times with tPBS(−), a chromogenicsubstrate, Peroxidase Substrate (Kirkegaad & Perry Laboratories), wasadded at 80 μL/well, followed by incubation for 20 minutes at roomtemperature. After the addition of Peroxidase Stop Solution (Kirkegaad &Perry Laboratories) at 80 μL/well, the absorbance at 405 nm was measuredwith a Microplate Reader Model 3550 (Bio-Rad Laboratories).

1-4 Purification of Antibody from Hybridoma Culture Supernatant

The resulting hybridomas described above were cultured in HAT selectivemedium using low-IgG FBS (Invitrogen) as the FBS. Protein G beads(Pharmacia), in which the solvent was replaced with wash buffer (20 mMsodium acetate buffer, pH 5.0), were added to 20-50 mL of the culturesupernatant at 50 μL per 10 mL of culture supernatant, followed bymixing by inversion overnight at 4° C. After the Protein G beads hadbeen retrieved and washed with wash buffer, the antibody was eluted withelution buffer (50 mM sodium acetate buffer, pH 3.3), followedimmediately by neutralization with neutralizing buffer (Tris-HCl buffer,pH 7.8). The buffer was replaced with phosphate-buffered physiologicalsaline (pH 7.35-7.65; Nissui Pharmaceutical) and the purified antibodywas concentrated using an ultrafiltration kit for a molecular weightfraction of 10 kDa (Amicon (registered trademark), Millipore), followedby sterilization with a 0.22 μm sterilization filter (Millipore GV,Millipore).

Example 2 Assay of Antibody-Induced Phosphorylation

The ability of the anti-AXL monoclonal antibody obtained in Example 1 toinduce the phosphorylation of AXL in cancer cells was tested. Cells(human non-small-cell lung cancer cell line Calu-1, human breast cancercell line MDA-MB-231, and human prostate cancer cell line DU-145) wereseeded into six-well plates at a density of 4×10⁵ cells/well and 24hours later, the medium was replaced with medium from which the serumhad been removed (serum-starved medium) and the cells were culturedovernight. Next, the above-prepared anti-AXL monoclonal antibody wasadded at 2 μg/mL, and recombinant GAS6 (R&D) was added at 200 ng/mL toact as the positive control, followed by incubation for 30 minutes at37° C. Next, the cells were washed with PBS(−) and lysed on ice for 30minutes with cell lysis buffer (137 mM NaCl, 20 mM Tris-HCl [pH 8.0],10% glycerol, 2 mM EDTA, 1 mM sodium vanadate, 1 vol % NP-40, 1 mMphenylmethylsulfonyl fluoride [PMSF], 10 μg/mL aprotinin, 10 μg/mLleupeptin, 10 μg/mL pepstatin). The cell solution mixture washomogenized with an ultrasonic homogenizer (Tomy Seiko) followed bycentrifugation (20,000×g) for 10 minutes at 4° C. The supernatant of thecell solution mixture was mixed for 30 minutes with 0.05 volumes ofProtein G Agarose (Roche Diagnostics). After centrifugation (2,300×g)for one minute at 4° C., 1.2 μg of anti-AXL monoclonal antibody (R&D)was added to the supernatant, which was shaken for one hour at 4° C.Then, 10 μL of Protein G Agarose was added and the solution was shakenfor a further one hour at 4° C. After centrifugation (2,300×g) for oneminute at 4° C., the immunoprecipitate was washed and suspended inNuPAGE-LDS sample buffer (Invitrogen), and then heated for 10 minutes at70° C. The immunoprecipitate was electrophoresed for one hour at 150 Vusing 7% NuPAGE (Invitrogen).

After immunoprecipitation and electrophoresis on 7% NuPAGE, the proteinwas electrophoretically transferred to a 0.45 μm polyvinylidenedifluoride filter (Immobilon-FL, Millipore) over the course of one hourat 30 mA with NuPAGE transfer buffer (Invitrogen) and the buffercontaining 20 vol % methanol. The filter was washed with TBS (50 mMTris-HCl [pH 7.6], 150 mM NaCl) and then blocked by incubation overnightin Odyssey blocking buffer (Li-COR). The filter was washed four timesfor five minutes each with TBST (TBS containing 0.05 vol % Tween(registered trademark) 20) and then incubated for two hours at roomtemperature with biotinylated 4G10 anti-phosphotyrosine antibody(diluted 1:1,000 with TBST; Upstate) and anti-AXL antibody (diluted1:15,000 with TBST; Santa Cruz). After the filter had been washed fourtimes for five minutes each with TBST, the filter was incubated for onehour with Alexa 680-labeled streptavidin (Invitrogen) diluted 1:10,000with TBST and IRDye 800-labeled anti-goat secondary antibody (Rockland)diluted 1:10,000 with TBST. After the filter had been washed three timesfor five minutes each with TBST, it was washed again once for fiveminutes with TBS, and then scanned with the Odyssey infrared imagingsystem (Li-COR).

A band obtained by immunoblotting the immunoprecipitated intracellularAXL with anti-AXL antibody and a band obtained by immunoblotting it withanti-phosphotyrosine antibody overlapped, and the intensification of theband for tyrosine-phosphorylated AXL was observed after the addition ofthe anti-AXL monoclonal antibodies Ax285, Ax292, Ax223, Ax96, and Ax258,and after the addition of the recombinant GAS6 used as the positivecontrol (FIGS. 1 a, b, c, d, and e). Thus, intensified tyrosinephosphorylation of AXL was observed as a result of the addition of theanti-AXL monoclonal antibody acquired by the present inventors. Thus,these anti-AXL monoclonal antibodies can induce the phosphorylation ofthe kinase domain of AXL.

Example 3 Assay of the Inhibition of Ligand-Dependent Phosphorylation bythe Antibody

The ability of the anti-AXL monoclonal antibody to inhibitligand-dependent phosphorylation within cancer cells was tested. Cells(human non-small-cell lung cancer cell line Calu-1, human breast cancercell line MDA-MB-231, or human prostate cancer cell line DU-145) wereseeded into six-well plates at a density of 4×10⁵ cells/well and 24hours later, the medium was replaced with medium from which the serumhad been removed (serum-starved medium) and then the cells were culturedovernight. Next, the anti-AXL monoclonal antibody prepared in Example 1was added at 2 μg/mL, and then recombinant GAS6 (R&D) was addedsimultaneously at 200 ng/mL and incubated for 30 minutes at 37° C. Next,the cells were washed with PBS(−) and the protein was extracted from thecells with the previously described cell lysis buffer. The cell lysisproducts, immunoprecipitated with commercially available anti-AXLantibody (Santa Cruz™), were separated on 7% NuPAGE (Invitrogen),immunoblotted by western blotting, and tyrosine phosphorylation assay,as previously described. The immunoprecipitated intracellular AXL wasblotted with anti-phosphotyrosine antibody by treatment with GAS6, whichis its ligand. However, the blot of the anti-phosphotyrosine antibodywas weakened by the anti-AXL monoclonal antibodies Ax7 and Ax51 (FIGS. 2a and b). Thus, the ligand-dependent tyrosine phosphorylation of AXL wasconfirmed to be inhibited by exposing to the anti-AXL monoclonalantibodies acquired by the present inventors. These anti-AXL monoclonalantibodies can inhibit ligand-dependent phosphorylation of the kinasedomain of AXL.

Example 4 Assay of the Induction of AXL Protein Downmodulation by theAntibody

The ability of the anti-AXL monoclonal antibody to induce thedownmodulation of AXL within cancer cells was tested. Cells (humannon-small-cell lung cancer cell line Calu-1, human breast cancer cellline MDA-MB-231, or human prostate cancer cell line DU-145) were seededinto six-well plates at a density of 4×10⁵ cells/well and 24 hourslater, the medium was replaced with medium from which the serum had beenremoved (serum-starved medium) and then the cells were culturedovernight. Next, the anti-AXL monoclonal antibody prepared as describedabove was added at 2 μg/mL, and recombinant GAS6 (R&D) was added at 200ng/mL to act as the positive control, followed by incubation for 24hours at 37° C. Next, the cells were washed with PBS(−) and the proteinwas extracted from the cells with the previously described cell lysisbuffer. The cell lysis products, immunoprecipitated with a commerciallyavailable anti-AXL antibody (Santa Cruz™), were separated on 7% NuPAGE(Invitrogen), immunoblotted by western blotting, and tyrosinephosphorylation assay, as previously described.

25 μg of each protein solution was suspended in NuPAGE-LDS sample buffer(Invitrogen), heated for 10 minutes at 70° C., and electrophoresed forone hour at 150 V on 7% NuPAGE (Invitrogen). The gels separated byelectrophoresis were electrophoretically transferred to a 0.45 μmpolyvinylidene difluoride filter (Immobilon-FL, Millipore) over thecourse of one hour at 30 mA in NuPAGE transfer buffer (Invitrogen) andthe buffer containing 20 vol % methanol. The filter was washed with TBS(50 mM Tris-HCl [pH 7.6], 150 mM NaCl) and then blocked by incubationovernight in Odyssey blocking buffer (Li-COR). The filter was washedfour times for five minutes each with TBST and then incubated for twohours at room temperature with anti-AXL antibody (diluted 1:15,000 withTBST; Santa Cruz) and anti-actin antibody (diluted 1:5,000 with TBST).After the filter had been washed four times for five minutes each withTBST, it was incubated for one hour with Alexa 680-labeled anti-rabbitsecondary antibody (Invitrogen) diluted 1:10,000 with TBST and IRDye800-labeled anti-goat secondary antibody (Rockland) diluted 1:10,000with TBST. After it had been washed three times for five minutes eachwith TBST, the filter was washed again once for five minutes with TBS,and then scanned with the Odyssey infrared imaging system (Li-COR).

The AXL blots were observed to weaken following exposure to the anti-AXLmonoclonal antibodies Ax285, Ax292, Ax223, Ax96, Ax258, Ax284, Ax7, andAx225 (FIGS. 3 a, b, c, d, e, f, g, and h). Therefore, these anti-AXLmonoclonal antibodies can induce the downmodulation of AXL protein.

Example 5 In Vitro Angiogenesis Inhibitory Activity of Anti-AXL Antibody

The activity of anti-AXL antibody to inhibit the lumen formation ofhuman umbilical vein endothelial cells (HUVEC) was measured using anangiogenesis kit available from Kurabo Industries. The experimentalprocedure was in accordance with the protocol provided with the kit andis summarized below. HUVEC and fibroblasts were cocultured, and a24-well plate (provided with the kit) containing cells in the growthstate of early lumen formation was placed in an incubator for threehours at 37° C. under 5% CO₂ and humidified air. The caps of threecontainers containing 25 mL of special-purpose medium (provided with thekit) were loosened and placed in the incubator for about 30 minutes at37° C. under 5% CO₂ and humidified air. The plate was then removed fromthe incubator and the well cap sheet was peeled off. The plate cover wasthen replaced with a new one (provided with the kit). The cells wereconfirmed to be normal by observation under a microscope. Culture medium(>12 mL/plate), warmed to 37° C., was dispensed into Falcon tubes andVEGF-A (2 μg/mL) was added to the medium to a final concentration of 10ng/mL by 200-fold dilution. The anti-AXL antibody prepared as describedabove was added to the medium dispensed into the tubes to a finalconcentration of 10 μg/mL. PBS(−) was used in place of antibody for thenegative control. The medium in the wells of the 24-well plate wasgently removed by aspiration and 500 μL of drug-containing medium wasthen gently added. The condition of the cells was observedmicroscopically and they were then returned to the incubator. The mediumwas replaced using the same procedure on days 4, 7, and 9, counting theday on which the antibody was added as day 1.

The cell layer was fixed and stained using a lumen staining kit (Kurabo)on the 11th day after the addition of the antibody. The procedure wascarried out according to the protocol provided with the kit and issummarized below. After the cells were observed under a microscope, themedium was removed by aspiration and the well was washed by the additionof 1 mL of wash buffer (PBS(−) pH 7.4; Sigma) to each well, and then thewash buffer was removed by aspiration. 1 mL of ice-cold fixing solution(70% ethanol) was added to each well and allowed to stand for 30 minutesat room temperature. The fixing solution was then removed, 1 mL ofblocking solution was added to each well, the well was washed, and theblocking solution was removed by aspiration. 0.5 mL of the primaryantibody provided with the kit was diluted according to the protocol andadded to each well, followed by incubation for one hour at 37° C. Theprimary antibody was removed by aspiration and each well was washedthree times with 1 mL of blocking solution (PBS(−) containing 1% BSA, pH7.4; Sigma). 0.5 mL of the secondary antibody provided with the kit anddiluted in accordance with the protocol was added to each well, followedby incubation for one hour at 37° C. The secondary antibody was removedby aspiration and each well was washed three times with 1 mL ofdistilled water. 0.5 mL of the substrate solution provided with the kitwas added to each well, followed by incubation for 10-30 minutes at 37°C. until the lumen became dark purple. The substrate solution was thenremoved by aspiration and each well was washed three times with 1 mL ofdistilled water and allowed to air dry. Microscopic images of each fixedwell were captured at five locations with a CCD camera (Nikon DigitalCamera, dxm1200), and the vessel areas were calculated usingangiogenesis quantification software (Ver. 1.0, Kurabo).

The rate of the reduction in the area of the vessels that formed in alumen in the wells to which was added anti-AXL antibody relative to thearea of the vessels that formed in a lumen in the wells to which wasadded the negative control PBS(−) was used as the index of theinhibitory activities of the antibodies, and Ax232, Ax292, Ax285, andAx284 displayed inhibitory activity (FIG. 4).

Example 6 Binding Activity of the Anti-AXL Antibody to Mouse AXL

After the extracellular domain of mouse AXL (hereinafter referred to asmAXL-ECD; R&D) was diluted with coating buffer (100 mM sodiumbicarbonate buffer, pH 9.6) to 2 μg/mL, 100 μL was dispensed into a96-well plate (Nunc-Immuno™ 96 MicroWell™ MaxiSorp™ plates; Nalge NuncInternational). After the plate was placed in a refrigerator overnight,the antibody solution in the plate was removed, 200 μL/well of diluentbuffer (BlockingOne; Nacalai Tesque) was dispensed, and then blocked fortwo hours at room temperature. After the removal of the diluent buffer,the anti-AXL antibody prepared above diluted to 3 μg/mL with diluentbuffer was dispensed at 100 μL/well, and allowed to stand for 1.5 hoursat room temperature. After the removal of the antibody solution, thewells were washed three times with tPBS(−). A labeled antibody cocktailcontaining alkaline-phosphatase-labeled goat anti-mouse IgG1 antibody,alkaline-phosphatase-labeled goat anti-mouse IgG2a antibody, andalkaline-phosphatase-labeled goat anti-mouse IgG2b antibody(SouthernBiotech) was prepared with final dilutions of each antibody of1/2250:1/4000:1/4000, and was dispensed at 100 μL/well, and allowed tostand for one hour at room temperature. After the removal of theantibody solution, the wells were washed three times with tPBS(−). 100μL/well of alkaline phosphatase chromogenic substrate solution (BluePhosMicrowell Phosphatase Substrate System, Kirkegaad & Perry Laboratories)was dispensed, followed by color development at room temperature. Theabsorbance at 650 nm was then measured with a microplate reader (Emax,Molecular Devices).

Binding to mouse AXL was confirmed for Ax96, Ax119, Ax223, Ax225, andAx284.

Example 7 In Vitro Cancer Cell Growth Inhibitory Activity of theAnti-AXL Antibody

Evaluation was performed using HCT-116 (CCL-247), Calu-1 (HTB-54),DU-145 (HTB-81), and T-47D (HTB-133) purchased from ATCC, and AsPC-1,MDA-MB-231, and PANC-1 purchased from Dainippon Sumitomo Pharma. Thecells were maintained under the conditions recommended by the supplierof each cell. A dilution series was prepared of the anti-AXL antibodyproduced as described above with 10% FBS/RPMI1640, and 20 μL wasdispensed into a 96-well plate (flat bottom). Each of the suspensions ofHCT-116, Calu-1, DU145, T-47D, AsPC-1, MDA-MB-231, and PANC-1 cells wereprepared at 2000, 3000, 2000, 5000, 3000, 5000, and 3,000 cells perwell, respectively, and 180 μL of cell suspension was added to each welland then cultured in an incubator at 37° C. in 5% CO₂. Four days later,10 μL of WST-8 (Cell Counting Kit-8, Dojindo Laboratories) was added toeach well and the absorbance at 450 nm was measured with a microplatereader (Model 3550-UV, Bio-Rad), according to the protocol provided withthe kit. The cell inhibitory activity (%) of the anti-AXL antibodies wascalculated by assigning a value of 0% inhibition to the value measuredwhen no test substance was included, and assigning a value of 100%inhibition to a value measured when no test substance or cells wereincluded.

Ax51 demonstrated CGI activity of 30% or more against HCT116 cells.

TABLE 2 HCT116 1st 2nd TOP ⅓ 1/9 TOP 1/10 Ax51 31 11 10 12 15

Example 8 Measurement of Antitumor Effects of the Anti-AXL Antibody in aMouse Model Grafted with Human Pancreatic Adenocarcinoma 1. Preparationof a Mouse Model Grafted With Human Pancreatic Adenocarcinoma

The human pancreatic adenocarcinoma cell line PANC-1, purchased fromDainippon Pharmaceutical (currently Dainippon Sumitomo Pharma), wasprepared at 5×10⁷ cells/mL with HBSS. 200 μL of the cell suspension(1×10⁷ cells/mouse) was subcutaneously grafted into the inguinal regionof a CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu (BALB-nu/nu) mouse purchased fromCharles River Laboratories, Japan. The mouse was subjected to theexperiment when the tumor volume had reached about 210 mm³.

2. Antibody Preparation and Administration

The antibodies of Table 1 were prepared at 2 mg/mL with PBS andadministered twice a week for two weeks at 20 mg/kg into the peritonealcavity of the mouse grafted with human pancreatic adenocarcinoma. As thenegative control, PBS was administered in the same manner. Gemzar (EliLilly Japan) was prepared at 12 mg/mL with physiological saline as thepositive control and administered intraperitoneally at 120 mg/kg twice aweek for two weeks.

3. Evaluation of Antitumor Effects

The antitumor effects in a mouse model grafted with human pancreaticadenocarcinoma were calculated as tumor-growth-suppressive effects bycomparing the tumor growth in the antibody-treated group with the tumorgrowth in the negative control group four days after the finaladministration (FIG. 5).

Tumor-growth-suppressive effect (%)=(1−amount of tumor growth in theantibody-treated group/amount of tumor growth in the control group)×100

4. Statistical Processing

Tumor volume was expressed as the mean±standard deviation. Statisticalanalysis consisted of a comparison between the control group and thetreated group by the LSD method using the SAS Preclinical Package Ver.5.0. Reliability of 95% (*: p<0.05) was determined to constitutesignificance.

5. Results

All of the antibodies inhibited tumor growth and demonstrated antitumoreffects (FIG. 5).

Example 9 Measurement of Antitumor Effects of Anti-AXL Antibody on MouseModel Transplanted with Human Pancreatic Adenocarcinoma (2)

1. Preparation of Mouse Model Grafted with Human PancreaticAdenocarcinoma

Human pancreatic adenocarcinoma cell line PANC-1 purchased fromDainippon Pharmaceutical (currently Dainippon Sumitomo Pharma) wasprepared to 5×10⁷ cells/mL with HBSS. 200 μL of the cell suspension(1×10⁷ cells/mouse) were subcutaneously grafted to the inguinal regionsof CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu (BALB-nu/nu) mice purchased fromJapan Charles River. The mice were used in the experiment when the meantumor volume reached about 270 mm³.

2. Antibody Preparation and Administration

Anti-AXL antibody was prepared to 2 mg/mL with PBS and administered intothe peritoneal cavity of the mice grafted with human pancreaticadenocarcinoma twice a week for two weeks at 20 mg/kg. PBS wasadministered in the same manner for use as a negative control. Gemzar(Eli Lilly Japan) was prepared to 12 mg/mL with physiological saline foruse as a positive control and administered intraperitoneally twice aweek for two weeks at 120 mg/kg.

3. Evaluation of Antitumor Effects

Antitumor effects in a mouse model grafted with human pancreaticadenocarcinoma were calculated as tumor growth suppressive effects bycomparing with the amount of tumor growth of a negative control groupfour days after final administration.

Tumor growth suppressive effect (%)=(1−amount of tumor growth of theantibody-treated group/amount of tumor growth of the control group)×100

4. Results

The results for suppression of tumor growth are shown in FIG. 6. A tumorgrowth suppressive effect (%) of lower than 30% is indicated as “−”,that of 30% or more is indicated as “+”, and that of 60% or more isindicated as “++”. The results for the assay of inhibition ofligand-dependent phosphorylation by antibody of Example 3 are also shownin FIG. 6.

Antibodies that bind to FND-1 demonstrated 60% or more of TGI activityeven if administration was begun at the time when mean tumor volumes hadreached about 270 mm³. This finding that anti-AXL antibodies that bindto FND1 have such significant antitumor effects in vivo was determinedfor the first time in this study and was completely unexpected.

In addition, the existence of anti-AXL antibodies that bind to IgD2 thatdemonstrate phosphorylation inhibitory effect and in vivo antitumoreffects as indicated in Examples 3, 8, and 9 was also discovered for thefirst time in this study and was also completely unexpected.

Example 10 Binding Activity to Human AXL-FND1 and Human AXL-IgD2 1.Binding Activity to Human AXL-FND1 and Human AXL-IgD2

The binding abilities of anti-AXL monoclonal antibody to AXL-fibronectintype 3 domain 1 (AXL-FND1) and AXL immunoglobulin family domain 2(AXL-IgD2) were tested.

2. Preparation of Human Recombinant AXL-FND1 and Human RecombinantAXL-IgD2 Expression Vectors

Human recombinant AXL-FND1 was prepared by amplifying by PCR a regionequivalent to the 225th to 331st amino acids from full-length human AXLcDNA (O'Bryan, et al., Mol. Cell. Biol. (1991) 11, 5016-5031) (GenBankNo. NM 021913), cloning the amplified products to pET-41a(+) (Novagen)to express fusion proteins with GST-tag, and constructing pET-AXL-FND1.Other domains were prepared by amplifying by PCR a region equivalent tothe 137th to 224th amino acids, and cloning the amplified products topET-41a(+) to express fusion proteins with GST tag. Each of the preparedvectors (5 μl) was transformed to DHSa (Toyobo Co., Ltd., Cat. No.DNA-903) by a heat shock method and then cultured in SOC medium.Colonies were selected after culturing overnight at 37° C. on an LBplate containing kanamycin.

3. Purification of Human Recombinant AXL-FND1 and Human RecombinantAXL-IgD2

Each of the produced colonies were precultured overnight at 37° C. in 20mL of LB medium containing kanamycin and then transferred to 500 mL ofmedium. The each colony was cultured to an A₆₀₀ of 0.5±0.05 and IPTG wasadded to be a concentration of 0.5 mM. After culturing for one hour at37° C., the bacterial cells were collected and suspended in Buffer A (50mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM PMSF, and 1 mM DTT). Freezingand thawing was repeated twice using liquid nitrogen. NP-40 was thenadded to 0.5% and the cells were homogenized with an ultrasonichomogenizer (30 seconds×5) and centrifuged for 30 minutes at 204,000×G,and then the supernatant was recovered.

Human recombinant AXL-FND1 was purified in the manner described belowusing the resulting supernatant. Solubilized E. coli supernatant wasmixed with Glutathione Sepharose™ 4 Fast Flow (GE Healthcare) andstirred for one hour at 4° C. with a rotator. After centrifugation forfive minutes at 500×G, the supernatant was discarded and the GlutathioneSepharose™ 4B was washed by adding Buffer A. This washing procedure wasrepeated three times. After transferring the human recombinant AXL-FND1from the washed Glutathione Sepharose™ 4 Fast Flow to a mini-column, itwas separated and eluted from the Glutathione Sepharose™ 4 Fast Flowwith 50 mM Tris-HCl (pH 7.5) and 25 mM glutathione. Each of other AXLdomains was expressed, separated, and eluted in the same manner.

4. Evaluation of Binding Activity of Anti-AXL Antibody to AXL-FND1 byWestern Blotting

The recombinant AXL-FND1 separated and eluted from the GlutathioneSepharose™ 4 Fast Flow, as well as AXL-IgD1, AXL-IgD2, AXL-FND2,AXL-IgD1+IgD2, AXL-IgD2+FND1, and AXL-FND1+FND2 were quantified usingthe BIO-RAD Dc Protein Assay. 1 μg each was mixed with NuPAGE® SampleBuffer (Invitrogen), and electrophoresed with NuPAGE® 10% Bis-TrisGel.The electrophoresed gel was transferred to an Immobilon™-FL (Millipore)PVDF membrane. The PVDF membrane containing the transferred protein wasblocked with Odyssey® Blocking Buffer (LI-COR) and immersed in a primaryantibody solution in which anti-AXL antibody was diluted to 5 μg/mL, andincubated overnight at 4° C. The PVDF membrane containing thetransferred protein and immersed in the primary antibody solution waswashed four times for five minutes each with 0.1% TBS-T (TBS(Tris-Buffered Saline (Takara)) containing 0.1% Tween-20). The PVDFmembrane immersed in anti-AXL antibody was immersed in Alexa Fluor® 680Goat Anti-mouse IgG (H+L) (Invitrogen) secondary antibody solutiondiluted to 80 ng/mL and incubated for one hour at room temperature.After washing the PVDF membrane immersed in the secondary antibodysolution three times for five minutes each with 0.1% TBS-T, the membranewas washed for five minutes with TBS-T containing 0.01% SDS and thenwashed for five minutes with TBS. The binding of the washed PVDFmembrane was then evaluated by scanning with the Odyssey® far infraredimaging system.

5. Results

The evaluation results are shown in FIG. 6.

Anti-AXL antibody produced by a hybridoma deposited under Accession No.FERM BP-10854 (Ax225) was demonstrated to recognize FND1 of AXL (FIG.6). Anti-AXL antibody produced by a hybridoma deposited under AccessionNo. FERM BP-10857 (Ax284) was considered to recognize FND1 and IgD2 ofAXL (FIG. 6). Anti-AXL antibody produced by a hybridoma deposited underAccession No. FERM BP-10850 (Ax7) and anti-AXL antibody produced by ahybridoma deposited under Accession No. FERM BP-10851 (Ax51) weredemonstrated to recognize IgD2 of AXL (FIG. 6).

Example 11 Measurement of Antitumor Effects of Anti-AXL Antibody onMouse Model Grafted with Human Breast Cancer

1. Preparation of Mouse Model Grafted with Human Breast Cancer

Human breast cancer cell line MDA-MB-435S obtained from ATCC wasprepared to 5×10⁷ cells/mL with HBSS. 200 μL of the cell suspension(1×10⁷ cells/mouse) was subcutaneously grafted to the inguinal regionsof CAnN.Cg-Foxn1<nu>/CrlCrlj nu/nu (BALB-nu/nu) mice purchased fromJapan Charles River. The mice were used in the experiment when the tumorvolume reached about 200 mm³.

2. Antibody Preparation and Administration

Anti-AXL antibody was prepared to 2 mg/mL with PBS and administered intothe peritoneal cavity of the mice grafted with human breast cancer twicea week for two weeks at 20 mg/kg. PBS was administered in the samemanner for use as a negative control.

3. Evaluation of Antitumor Effects

Antitumor effects in a mouse model grafted with human breast cancer werecalculated as tumor growth suppressive effects by comparing with theamount of tumor growth of a negative control group four days after finaladministration.

Tumor growth suppressive effect (%)=(1−amount of tumor growth of theantibody-treated group/amount of tumor growth of the control group)×100

4. Statistical Processing

Tumor volume was expressed as the mean±standard deviation. Statisticalanalysis consisted of a comparison between the control group and thetreated group by the LSD method using the SAS Preclinical Package Ver.5.0. Reliability of 95% (*: p<0.05) was determined to constitutesignificance.

5. Results

The used anti-AXL antibodies suppressed tumor growth and demonstratedantitumor effects (FIG. 7). Therefore, anti-AXL antibodies that bind toFND1 are expected to have antitumor effects against various tumors.

Example 12 Sequence Analysis of Antibody cDNA

1. Preparation of chimeric antibody-expression vectors

Total RNA was extracted from the cells of a hybridoma deposited underAccession No. FERM BP-10854 (Ax225) using the RNeasy Mini Kit (Qiagen),and cDNA was synthesized using the SMART RACE cDNA Amplification Kit (BDBiosciences). Antibody variable region gene was isolated by carrying outPCR with PrimeSTAR HS DNA Polymerase (Takara) using the followingprimers (H chain, MHCg1; L chain, MLCk) which were set for respectiveconstant regions of antibody and 10× Universal Primer A Mix, providedwith the SMART RACE cDNA Amplification Kit (BD Biosciences).

MHCg1: 5′-GGGCCAGTGGATAGACAGATG-3′ (SEQ ID NO. 1) MLCk:5′-GCTCACTGGATGGTGGGAAGATG-3′ (SEQ ID NO. 2)

The nucleotide sequence of each isolated DNA fragment was determinedusing the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems)with the ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer(Applied Biosystems) in accordance with the method described in theinstructions provided.

2. Results

The heavy chain variable region of the amino acid sequence of theresulting AXL225 mouse antibody is shown in SEQ ID NO. 3, the CDR1 ofthat region is shown in SEQ ID NO. 4, CDR2 is shown in SEQ ID NO. 5, andCDR3 is shown in SEQ ID NO. 6. The light chain variable region of theamino acid sequence of the resulting AXL225 mouse antibody is shown inSEQ ID NO. 7, the CDR1 of that region is shown in SEQ ID NO. 8, CDR2 isshown in SEQ ID NO. 9, and CDR3 is shown in SEQ ID NO. 10.

INDUSTRIAL APPLICABILITY

The present inventors discovered for the first time that anti-AXLantibodies have an angiogenesis-suppressing effect and acancer-suppressing effect. The anti-AXL antibody of the presentinvention is useful as an angiogenesis inhibitor and as a cell-growthsuppressant. Using an antibody of the present invention, thephosphorylation of AXL can also be induced or inhibited. Moreover, usingan antibody of the present invention, the expression level of AXL can bereduced.

1.-46. (canceled)
 47. A monoclonal antibody that binds to fibronectintype III domain 1 (FND1) of human anexelekto (AXL), hascell-growth-suppressive activity, and does not inhibit phosphorylationof AXL.
 48. The antibody according to claim 47, wherein the antibodysuppresses cancer cell growth.
 49. The antibody according to claim 47,wherein the antibody has an activity that reduces AXL expression level.50. The antibody according to claim 47, wherein the antibody hasangiogenesis inhibitory activity.
 51. The antibody of claim 47, whereinphosphorylation of AXL is induced by the binding of an AXL ligand toAXL.
 52. The antibody of claim 48, wherein phosphorylation of AXL isinduced by the binding of an AXL ligand to AXL.
 53. A monoclonalantibody prepared using as an antigen a peptide consisting of the entireamino acid sequence of FND1 of human AXL or an amino acid sequencecomprising five or more consecutive amino acids thereof, wherein theantibody has cell-growth-suppressive activity and does not inhibitphosphorylation of AXL.
 54. The antibody according to claim 53, whereinthe antibody has an activity that reduces AXL expression level.
 55. Theantibody according to claim 53, wherein the antibody has angiogenesisinhibitory activity.
 56. The antibody of claim 53, whereinphosphorylation of AXL is induced by the binding of an AXL ligand toAXL.
 57. A monoclonal anti-AXL antibody that competes for binding to thesame epitope bound by either of (a) a monoclonal antibody (Ax284)produced from a hybridoma deposited with the IPOD, AIST, and assignedAccession No. FERM BP-10857; or (b) a monoclonal antibody (Ax225)produced from a hybridoma deposited with the IPOD, AIST, and assignedAccession No. FERM BP-10854, wherein the anti-AXL antibody blocks thebinding of either Ax284 or Ax225 to AXL protein by at least 50% ascompared to a control level of binding of either Ax284 or Ax225 to AXLprotein in the absence of the anti-AXL antibody.
 58. A method ofinhibiting angiogenesis in a subject, the method comprisingadministering to a subject in need thereof the antibody according toclaim
 47. 59. A method of suppressing cell-growth in a subject, themethod comprising administering to a subject in need thereof theantibody according to claim
 47. 60. The method of claim 59, wherein theantibody suppresses growth of a cancer cell.
 61. A method of treatingcancer in a subject, the method comprising administering to a subject inneed thereof the antibody according to claim
 47. 62. The method of claim61, wherein the cancer is glioma, gastric cancer, endometrial cancer, ornon-small-cell lung cancer.
 63. The method of claim 61, wherein thecancer is pancreatic cancer, gastric cancer, lung cancer, osteosarcoma,colon cancer, prostate cancer, melanoma, endometrial cancer, ovariancancer, uterine leiomyoma, thyroid cancer, cancer stem cell, breastcancer, bladder cancer, renal cancer, glioma, neuroblastoma, oresophageal cancer.
 64. The method of claim 63, wherein the cancer ispancreatic adenocarcinoma or breast cancer.
 65. A method for reducing alevel of AXL protein expression in a cell, the method comprisingcontacting a cell that expresses AXL protein with the antibody accordingto claim
 47. 66. The method of claim 65, wherein the cell is contactedwith the antibody in vitro.
 67. The method of claim 65, wherein the cellis contacted with the antibody in vivo.